Method for extending pregnancy in patients exhibiting at least one symptom of preeclampsia and eclampsia

ABSTRACT

A composition is provided to prevent, limit the effects of, delay the onset of, or treat one or more of the causes, symptoms or complications of gestational hypertension, preeclampsia, eclampsia and/or intrauterine growth restriction. The composition comprises a therapeutically effective amount of an antibody that reacts immunologically with or binds digoxin and has a high dose of digoxin binding capacity as the active ingredient. There is also provided a method of preventing, limiting the effects of, delaying the onset of, or treating a cause, symptom or complication of gestational hypertension, preeclampsia, eclampsia or intrauterine growth restriction, comprising the step of administering to a mammal a composition comprising a therapeutically effective amount of an antibody that reacts immunologically with or binds digoxin and has a high dose of digoxin binding capacity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and is a continuation in part ofco-pending U.S. application Ser. No. 10/202,957, filed Jul. 25, 2002,and U.S. application Ser. No. 10/292,338, filed Nov. 12, 2002, andprovisional U.S. Application No. 60/681,693, filed May 17, 2005, each ofwhich applications is incorporated herein by this reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

APPENDIX

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of medicine and,more particularly, to prevention and treatment of diseases or conditionsassociated with elevated levels of endogenous sodium pump inhibitors,including, without limitation, the pregnancy-related conditions known asgestational hypertension, preeclampsia, eclampsia and intrauterinegrowth restriction.

2. Related Art

Conception results from the fertilization of an egg by a sperm and thedevelopment of the resulting embryo into a fetus. In order for pregnancyto be established and the embryo to develop it must embed itself withinthe uterine wall. At about 12 weeks' gestation a temporary disk-shapedorgan forms (the placenta), enhancing the transfer of oxygen andnutrients to, and permitting the removal of waste products from, thefetus. The placenta is critical to fetal development, and improperplacental formation is associated with preeclampsia and intrauterinegrowth restriction.

Upon conception, the fertilized egg (embryo) undergoes repeated celldivision and cell migration to form a blastocyst, a single layer ofcells surrounding a central cavity. One area of the blastocyst wall thatis three or four cells thick, known as the embryonic pole, becomesrecognizable as the embryo and eventually develops into the fetus. Theremaining blastocyst cells form a structure called the trophoblast.Pregnancy begins upon implantation of the blastocyst. Implantationoccurs when the trophoblasts proliferate and invade the uterine wall sothat the blastocyst burrows into the central layer of tissue(endometrium). The trophoblasts then develop to form the chorion (outermembrane) and amnion (inner membrane) surrounding the embryo. Theamniotic sac fills with fluid and expands to envelop the embryo. Theembryo continues to grow but is confined within one wall of the uterusuntil about the 12th week of gestation. At that time, the endometriumtissue overlying the embryo comes in such close contact with the tissueof the opposite uterine wall that they fuse and obliterate theendometrial cavity. The only cavity that remains in the uterus is theamniotic cavity, containing amniotic fluid and the fetus.

Placentation begins at about 10 days' gestation, when the trophoblastsinvade the endometrium and its blood vessels (spiral arterioles). Asearly as day 11 or 12, branch-like cell formations (villi) begin to formon the chorionic surface. Invasion of the maternal spiral arteriolescauses maternal blood to leak into spaces between the villi, providingnourishment to the developing embryo. At about 12 weeks' gestation, theplacenta begins to form as a distinct, disk-shaped organ. The placentais attached by the villi to the decidua directly overlying maternalspiral arterioles. The maternal spiral arterioles empty maternal bloodinto the intervillous space so that the blood circulates around andthrough the latticework of villi. Nutrients are transferred frommaternal blood in the intervillous space, across trophoblast cells,through the fibrous core of the villus, and through the endothelialcells of the fetal capillaries to the fetal blood. Fetal wastes move inthe opposite direction. The placenta reaches its final development atapproximately 18 to 20 weeks' of pregnancy.

It is generally known that abnormal placentation and placental vascularinsufficiency are central features of certain pregnancy-related medicalconditions, including, without limitation, preeclampsia and intrauterinegrowth restriction. Preeclampsia is a rapidly progressive condition,characterized by the occurrence of high blood pressure and abnormallevels of protein in the urine (proteinuria). Eclampsia is a more severeform of preeclampsia that is also characterized by seizures. Gestationalhypertension is hypertension in pregnancy without proteinuria, and itmay be a less severe form of or a precursor to preeclampsia.Preeclampsia and gestational hypertension may be further classified asmild or severe depending upon the severity of the clinical symptoms.These hypertension-related disorders are collectively referred to hereinas “pregnancy-induced hypertension” or “PIH.”

Typically, clinical symptoms of PIH occur in the late second trimesteror in the third trimester of pregnancy, although symptoms may occurearlier in pregnancy. PIH may be superimposed over other forms ofhypertension, such as essential and secondary hypertension, that existprior to or develop early in pregnancy. An increased risk for PIH isassociated with first time pregnancies, when there is a large intervalbetween pregnancies, pregnant women under the age of 20 or over the ageof 35, women of black race, multi-gestational pregnancies, women whohave conceived through in vitro fertilization (“IVF”), women who havehad a prior pregnancy with PIH, women who have had a prior pregnancyconceived with a different partner, women with a family history of PIHor high blood pressure or diabetes, women who are of higher than normalweight or body mass index prior to pregnancy, undernutrition, women witha personal history of polycystic ovarian syndrome, insulin resistence ordiabetes, hypertension, renal (kidney) disease, rheumatoid arthrisis,systemic lupus erythematosus or other autoimmune diseases, orthrombophilia risk factors. The risk of recurrent PIH in subsequentpregnancies is approximately thirty-three percent (33%), and PIH issuperimposed in twenty-five percent (25%) of pregnancies in whichchronic hypertension is present before pregnancy.

It is believed PIH occurs in five percent (5%) to ten percent (10%) ofall human pregnancies. PIH disorders are a leading global cause ofmaternal and infant illness and death. PIH occurs in over six millionbirths a year and is responsible for 15 percent of all premature births.By conservative estimates, these disorders are responsible for 76,000deaths each year. The risk of death for a pregnant woman with severepreeclampsia is 0.5%, and the risk of perinatal death for her baby is13%; if the condition remains untreated and eclampsia develops, the riskof maternal and fetal death increases to 5% and 28%, respectively. ZupanJ., Perinatal Mortality in Developing Countries, New Engl J. Med.352(20) 2047-248 (2005).

PIH is a syndrome having maternal and fetal manifestations. The maternalcondition is characterized by vasospasm, activation of the coagulationsystem, oxidative stress and inflammatory-like responses, all of whichhave detrimental effects on the placenta, kidney, blood, liver,vasculature, cardiopulmonary system and brain. PIH is a systemicsyndrome, and several of its non-hypertensive symptoms and complicationsmay be life-threatening even with only mild increases in blood pressure.

Intrauterine growth restriction or retardation (“IUGR”) and intrauterinefetal demise are fetal symptoms of or complications associated with PIH.IUGR is the second leading cause of perinatal morbidity and mortality,and it occurs in approximately 5% of the general obstetric population.Placental insufficiency, preeclampsia and abnormal placentation aregenerally recognized by those skilled in the art as being among thecauses of or contributory factors in IUGR.

Women who develop mild gestational hypertension after 37 weeks'gestation have pregnancy outcomes similar to those of pregnant women whoare normotensive, apart from increased rates of induced labor andcesarean delivery. Conversely, women with severe gestationalhypertension have high rates of placental abruption, preterm deliveryand small-for-gestational-age babies (the postnatal counterpart andlikely result of IUGR) similar to those of women with severepreeclampsia. Twenty-five percent (25%) of cases of eclampsia occurpostpartum, usually in the first 2 to 4 days after delivery.

After a diagnosis of severe PIH, the baby is generally induced anddelivered if it is near term, i.e., after 36 weeks. However, if PIHoccurs earlier in the pregnancy, its impact is more profound becausefetal viability is low; infant death occurs in approximately 87% ofthese cases. For pregnancies in which PIH occurs earlier than 24 weeks,the induction of labor is recommended and results in essentially 100%neonatal mortality. For pregnancies between 24 and 28 weeks' gestation,management of PIH may be attempted to increase gestational age, providedthat there is close monitoring for maternal and fetal complications.Regardless of gestational age of the fetus, delivery is the managementmethod of choice for eclampsia.

To date, there is no cure or effective treatment for PIH or IUGR.Delivery of the baby and placenta usually resolves the maternal symptomsof PIH within twelve (12) weeks' postpartum. However, if the baby is notnear term then early delivery is generally contrary to the bestinterests of the baby. Prophylactic measures against PIH, includingcalcium supplementation, vitamin and antioxidant supplementation andaspirin therapy, have not proven to be successful.

Depending upon the stage of the pregnancy and the severity of maternaland fetal conditions, gestational hypertension, preeclampsia, eclampsia,and IUGR may be managed in an attempt to prolong the pregnancy andadvance the gestational age to improve the fetal outcome. If maternalsymptoms persist after delivery, management of PIH symptoms is necessaryto prevent deterioration of the maternal condition or furtherdevelopment of complications.

Traditional management of PIH includes bed rest and antihypertensiveand/or anticonvulsant therapy, including, without limitation,hydralazine, nifedipine, sodium nitroprusside, 1-methyldopa (e.g.,Aldomet®), atenolol, labetalol, magnesium sulfate and phenytoin.Management of IUGR includes treatment of the concomitant maternaldisease (e.g., gestational hypertension or preeclampsia) and bed rest.Preterm delivery may be necessary to prevent intrauterine demise due tochronic fetal oxygen deprivation or if the maternal condition does notrespond to management efforts.

The cause(s) of PIH and IUGR remain elusive. The extensive list ofpossible causes currently being investigated by those skilled in the artinclude (1) immunologic factors (maternal reaction to paternal antigenor circulating auto-antibodies that activate angiotensin II), (2)genetic factors, (3) insulin resistance and increased levels of insulin,free fatty acids and triglycerides, (4) dietary calcium deficiency, (5)increased oxidative stress, (6) prostaglandin imbalance (increased ratioof thromboxane to prostacyclin), and (7) circulating pro-angiogenicfactors and their inhibitors (e.g., soluble fms-like tyrosine kinase 1,an agonist of vascular endothelial growth factor and placental growthfactor). Roberts J M and Gammill H S, Preeclampsia Recent Insights,Hypertension (December 2005) 1243-1249; Noris M, et al., Mechanisms ofDisease: Preeclampsia, Nature of Clinical Practice Nephrology 1(2):98-110 (December 2005); Solomon C G and Seely E W,Preeclampsia—Searching for the Cause, N. Engl. J. Med. 350(7):641-642(2004); Davison, J M, et al., New Aspects in the Pathophysiology ofPreeclampsia, J. Am. Soc. Nephrol. 15:2440-48 (2004); Pridjian G andPuschett J B, Preeclampsia Part 2: Experimental and GeneticConsiderations, Obstet. Gynecol. Survey 57(9):619-634 (2002)(summarizingearly research to determine the role of endogenous digitalis-likefactors in preeclampsia and concluding that results are unclear).

Notwithstanding the state of the art and various popular theories beinginvestigated by others in the field, it is theorized by Applicant thatcertain endogenous “digoxin-like” factors originating from maternal,placental and/or fetal sources may be a cause of or a contributingfactor in these conditions. Serum of adult patients in renal or liverfailure, pregnant women, neonates and umbilical cord blood evidenceendogenous factors that cross-react with anti-digoxin antibodies whenassayed with commercially available immunoassays for digoxin. Some, butnot all, studies have shown that these endogenous factors are present athigher levels in women with preeclampsia than in women withoutpreeclampsia.

These endogenous factors have generally been referred to as endogenous“digoxin-like,” “digitalis-like,” “endoxin,” “endobain,” “sodium pumpinhibitors” or “sodium pump ligands” not only because they cross-reactwith digoxin antibodies, but because they are also known to inhibitactivity of sodium/potassium ATPase in vitro. Certain known exogenoussodium pump inhibitors belong to classes of compounds known ascardenolides and bufadienolides, commonly referred to as cardiotonicsteroids or cardiac glycosides. The aglycone moieties of cardenolidesand bufadienolides are also known to be sodium pump inhibitors. Pullen MA, et al., Characterization of the Neutralizing Activity ofDigoxin-Specific Fab Toward Ouabain-like Steroids, J. Pharm. and Exp.Therapeutics 310(10): 319-325 (2004).

For the better part of a generation, the possible role of theseendogenous factors in PIH and IUGR has been generally discounted ordiscredited by leading investigators in the field, who found that therewas no difference between levels of endogenous factors in women with andwithout preeclampsia and who, therefore, concluded that endogenousfactors are not predictive of and do not play a major role inpreeclampsia. See, Gonzales, A R, et al., Digoxin-like ImmunoreactiveSubstance in Pregnancy, Am. J. Obstet. Gynecol. 157(3):660-664 (1987);Phelps, S J, et al., The Influence of Gestational Age and Preeclampsiaon the Presence and Magnitude of Serum Endogenous Digoxin-likeImmunoreactive Substance(s), Am. J. Obstet. Gynecol. 158(1): 34-39(1988). Thus, endogenous factors are not among the models of PRI or IUGRbeing currently suggested or investigated by other researchers generallyskilled in the art. Roberts J M and Gammill H S, Hypertension (December2005) 1243-1249; Noris M, et al., Nature of Clinical Practice Nephrology1(2): 98-110 (December 2005); Solomon C G and Seely E W, N. Engl. J.Med. 350(7):641-642 (2004); Davison, J M, et al., J. Am. Soc. Nephrol.15:2240-48 (2004); Redman C W, Sargent I L, Latest Advances inUnderstanding Preeclampsia, Science 308(5728):1592-94 (2005); Pridjian Gand Puschett J B, Obstet. Gynecol. Survey 57(9):619-634 (2002).

Applicant has discovered that symptoms of PIH and IUGR may be due, inwhole or in part, to inhibition of the sodium pump by endogenousfactors. It is theorized by Applicant that a decrease in sodium pumpactivity, particularly in vascular endothelial cells, may cause anincrease in intracellular sodium and calcium ions, promotingvasoconstriction, vasospasm and the resultant hypertension found in PIH.Furthermore, in placental cells, many nutrient transport processes arecoupled to Na⁺ transport and energized by the Na⁺ gradient. Thus,Applicant believes that inhibition of the sodium pump by theseendogenous factors may also impair nutrient and oxygen supply to theplacenta, restrict nutrient and blood flow to the developing fetus, andlimit the removal of metabolic waste products from the fetus—all ofwhich cause or contribute to IUGR.

It is known that, in vitro, sodium pump inhibition by endogenous factorsmay be reversed or prevented by addition of antibodies to cardenolidesand bufadienolides, particularly digoxin immune Fab. Pullen M A, et al.,J. of Pharm. and Exp. Therapeutics 310(10): 319-325 (2004). As disclosedin the prior and co-pending U.S. application Ser. Nos. 10/202,957,10/292,338, and 60/681,693, Applicant has discovered that antibodies tocardenolides and bufadienolides are useful in diagnosing and/or treatingthe causes, symptoms and/or complications of PIH and IUGR.

Determination of an effective antibody composition requires, inter alia,a determination of the body load of antigen that must be neutralized bythe antibody. There is no known available immunoassay specific for theendogenous factors that Applicant believes cause or contribute to PIHand IUGR. However, commercially available immunoassays have beendeveloped to detect digoxin, ouabain and marinobufagenin. Commerciallyavailable immunoassays for exogenous cardiac glycosides (particularly,immunoassays for digoxin, oubain and marinobufagenin) that have beenused to detect serum levels of endogenous factors in pregnant women withand without preeclampsia, have detected the following levels ofendogenous factors: <0.1 to 1.5 ng/mL, “digoxin”; 0.54 to 0.86 nmol/L,“ouabain”; and 2.53 to 2.73 nmol/L “marinobufagenin.” Lopatin D A, etal., Circulating Bufodienolide and Cardenolide Sodium Pump Inhibitors inPreeclampsia, J. Hypertension 17(8): 1179-1187 (1999); Adair C D, etal., Elevated Endoxin-Like Factor Complicating a Multifetal SecondTrimester Pregnancy: Treatment with Digoxin-Binding Immunoglobulin, Am.J. Nephrol. 16:529-531 (1996); Seely E W, et al., Markers of Sodium andVolume Homeostasis in Pregnancy-Induced Hypertension, J. Clin.Endocrinol. Metabol. 74(1): 150-156 (1992); Craig H R, et al., Bindingof Endogenous Digoxin-like Immunoreactive Factor to Serum ProteinsDuring Normal and Hypertensive Pregnancy, J. Clin. Immunoassay 14(4):245-250 (1991); Goodlin R C, Antidigoxin Antibodies in Eclampsia, N.Engl. J. Med. 618(8): 518-519 (Feb. 25, 1988); Goodlin R C, WillTreatment with Digoxin Antibody Benefit Pregnant Patients with Toxemiaand Elevated Digoxin Like Factor?, Medical Hypothesis 24:107-110 (1987);Beyers A D, et al., The Possible Role of Endogenous Digitalis-likesubstance in the Causation of Pre-eclampsia, SA Medical Journal, 65:883-885 (1984); Gusdon J P, et al., A Digoxin-like ImmunoreactiveSubstance in Preeclampsia, Am. J. Obstet. & Gynecol. 150:83 (1984);Graves S W and Williams G H, An Endogenous Ouabain-like FactorAssociated with Hypertensive Pregnant Women, J. Endocrinol. Metab.59:1070 (1984).

However, it is also known that there is variation among immunoassays indetecting endogenous “digoxin-like” factors. Furthermore, a number ofsubstances such as steroids, lipids and bile are known to cross-reactwith anti-digoxin antibodies and may interfere with detection ofendogenous factors in patients who have not been treated with digoxin ordigitalis. Ghione S, et al., Endogenous Digitalis-like Activity in theNewborn, J. Cardio. Pharmacol. 22: S25-S28 (1993); McMillan G A, et al.,Comparable Effects of Digibind and DigiFab in Thirteen DigoxinImmunoassays, Clin. Chemistry 48(9): 1580-84 (2002); Pudek M R, et al.,Seven Different Digoxin Immunoassay Kits Compared with Respect toInterference by a Digoxin-Like Immunoreactive Substance in Serum fromPremature and Full Term Infants, Clin. Chem. 29(11): 1972-1974 (1983).

Thus, Applicant believes that immunoassays specific for exogenouscardenolides or bufadienolides, including digoxin immunoassays, have notaccurately detected the levels of endogenous factors in pregnantpatients. It is believed that a substantial portion of endogenousfactors, perhaps up to 90%, are protein-bound and not detectable bydirect measurement with conventional immunoassay techniques. Valdes R,Graves S W, Protein binding of Endogenous Digoxin-immunoactive Factorsin Human Serum and its Variation with Clinical Condition, J. Clin.Endocrinol. Metabol. 60:1135-1143 (1985). This is further evidencedbecause sodium pump inhibition by endogenous factors substantiallyexceeds that which would be expected based upon the levels of endogenousfactors detected by conventional immunoassay. Pullen M A, et al., J. ofPharm. and Exp. Therapeutics 310(10): 319-325 (2004).

The discrepancy in immunoassay measurements of endogenous factors andlack of concordance with sodium pump inhibition suggests that there aredifferences between endogenous factors and exogenous cardenolides andbufadienolides. Miyagi H, et al., Ouabain-like Na/K-ATPase InhibitoryActivity of a Plasma Extract in Normal Pregnancy and Pregnancy InducedHypertension, Japan. J. Pharmacol. 57: 571-581 (1991). Thus, it istheorized by Applicant that the endogenous factors are not digoxin,ouabain, bufalin, marinobufagenin or other known exogenous cardenolidesand bufadienolides, but are one or more compounds that differ inbiological, chemical, physical, biopharmaceutical and/or pharmacokineticcharacteristics from exogenous cardiac glycosides.

Applicant has discovered that if antibodies to exogenous cardenolidesand bufadienolides are to be useful in diagnosing, preventing and/ortreating PIH and IUGR, an effective antibody composition may not bedetermined solely upon measurements of endogenous factors resulting fromimmunoassays specific for exogenous cardiac glycosides, such as digoxin,ouabain, bufalin or marinobufagenin.

Except as described in related U.S. application Ser. Nos. 10/202,957,10/292,338, and PCT/US2003/023235, WO 2004/011028 A1 (each of whichapplications is incorporated herein by this reference), there is noknown efficacious composition of antibodies that bind digoxin (includingany other exogenous cardenolide or bufadienolide that is not specificfor digoxin), or method of using such antibody compositions, forpredicting, preventing, diagnosing or treating gestational hypertension,preeclampsia, eclampsia or intrauterine growth restriction.

Goodlin (1988) and Adair, et al. (1996) have investigated the effects ofdigoxin antibodies on preeclampsia in vivo. These investigatorsadministered antibody compositions based upon measured serum digoxinconcentrations, doses and standard dosing formulas for treating digoxinintoxication. These initial experiments failed to establish digoxinantibody compositions that were effective for treating the symptoms ofpreeclampsia, extending pregnancy or advancing fetal development.

In the first experiment, Goodlin intravenously administered 10 mg totaldigoxin antibodies to a preeclamptic patient having a serum endogenousfactor level of 0.3 ng/mL (as determined by digoxin immunoassay). The 10mg composition was repeated once after 12 hours. Each antibodycomposition administered to the patient produced a precipitous, albeittransient, reduction in mean blood pressure. The patient's bloodpressure began to rise approximately one hour after each antibodycomposition was administered. Goodlin did not report the reduction inmean blood pressure to be statistically significant. Furthermore, theresults cannot be attributed solely to administration of digoxinantibodies because of the concurrent intravenous administration ofantihypertensive drugs and albumin. Goodlin states that the increase inurinary output was due, in part, to concurrent administration ofalbumin. However, Goodlin did not address the synergistic or combinedeffects of the antihypertensive drug administration in combination withdigoxin antibodies. Regrettably, and most significantly from ascientific and medical perspective, the pregnancy was terminatedprematurely and the fetus did not survive. Goodlin R C, New Engl. J.Med. 618(8): 518-519 (1988).

In the second experiment, Adair, et al., administered a singlecomposition of 29 mg total digoxin antibodies to a patient (twingestation) exhibiting a serum endogenous factor level of 0.4 ng/mL (asdetermined by digoxin immunoassay). The composition was given as apartial bolus (5 mg) and a slow infusion at a rate of 1 mg/hour for 24hours. Although mean arterial pressure gradually declined untilapproximately 12 hours after the treatment commenced, the reduction wasnot statistically significant. Moreover, during digoxin antibodytreatment the patient exhibited more than a two-fold increase inproteinuria. The worsening proteinuria led to premature termination ofthe pregnancy and, as in Goodlin, neither of the fetuses survived. AdairC D, et al., Am. J. Nephrology 16:529-531 (1996). Thus, neither Goodlinnor Adair established an antibody composition that was therapeuticallyeffective for treating preeclampsia.

Endogenous factors may also cross-react with antibodies specific forother non-digoxin cardenolides or bufadienolides, such as bufalin,ouabain or marinobufagenin. PCT US 2004/002802, WO 2004/071273 A2(republished WO 2004/071273 A3) (incorporated herein by this reference),claims that increasing urinary levels of marinobufagenin are adiagnostic indicator of preeclampsia. This patent application has alsogenerally suggested that antibodies to marinobufagenin may be used totreat preeclampsia. However, patent application WO 2004/071723 does notteach a therapeutically effective composition or dosing administrationregimen for treating preeclampsia with marinobufagenin antibody.

To date, PIH is believed to be a condition specific to humans. Thus,there is no known naturally occurring animal model of PIH available forthe study of gestational hypertension, preeclampsia or eclampsia.Recently, some investigators have attempted to create an animal model byadministering a high salt diet to pregnant rats and suggesting thatpregnant rats on the high salt (NaCl) diet exhibit symptoms of“preeclampsia.” It has been shown that an antibody to marinobufageninlowers blood pressure in the pregnant rats on high NaCl intake. Fedorovaet al., Antibody to Marinobufagenin Lowers Blood Pressure in PregnantRats on a High NaCl Intake, J. Hypertension 2005: 23(4):835-842.However, this proposed animal model has substantial differences fromnaturally occurring preeclampsia in humans. For example, the manner oftrophoblast invasion and placentation are significantly differentbetween rats and humans. The mild increase in urinary protein in therats does not approach significant proteinuria (>300 mg/24 hours) thatevidences preeclampsia in humans. Furthermore, the results of theantibody “treatment” are suspect because the treatment was with wholeantibody and not Fab fragments. Therefore, the resulting reduction inblood pressure in rats may be due, in whole or in part, to an allergicor immune reaction to whole antibody instead of a “binding-inactivation”of the endogenous factor by the antibody. Another major difficulty indesigning or evaluating a study involving an animal model ofpreeclampsia relates to the manipulation and intervention to which theanimal is subjected. This includes a number of factors such as thehandling of the animals, the route of administration of any agent, andthe methods used to measure the different variables, particularly bloodpressure. Most methods for measuring blood pressure require restrainingor tethering the animal which could lead to artificial elevation ofblood pressure, especially in cases where compensatory mechanisms havebeen eliminated. The effect of stress in the animals caused by therequired manipulation, in and of itself, has been clearly shown toresult in changes which could mimic preeclampsia. Thus, this recentlyproposed animal model has not proven to be an accurate model of truepreeclampsia and has not been generally accepted for the study ofpreeclampsia.

Applicant has disclosed in related U.S. application Ser. No. 10/202,957that administering anti-digoxin antibodies to preeclamptic womenmitigates or reverses the symptoms or complications of PIH and IUGR. Itis believed that by mitigating or reversing the symptoms orcomplications of PIH, PIH and IUGR will be controlled, the pregnancy maybe extended and fetal development may be advanced. Also, in related U.S.application Ser. No. 10/292,338 and PCT/US2003/023235, WO 2004/011028 A1it has been further disclosed that antibodies to digoxin may be used tocontrol or regulate, inter alia, Na⁺/K⁺ ATPase, to improve maternalblood flow, nutrient exchange and metabolic waste removal between thematernal vasculature and the placenta and fetus, and to prevent or limitIUGR.

The present invention is directed to overcoming one or more of theproblems set forth above, including overcoming the lack of apharmaceutical composition that is effective for preventing or treatingone or more causes, symptoms or complications of PIH or IUGR, that doesnot have adverse side effects, and that prolongs a PIH or IUGR pregnancyto allow further development of the fetus. It would be particularlybeneficial for pregnancy to be prolonged for a period of time sufficientto administer therapeutically effective doses of corticosteroids orother pharmaceutical compositions that either advance fetal organdevelopment or that prevent or limit the adverse physical consequencesin the neonate that may be due, in whole or in part, to prematuredelivery. It would also be beneficial to have a pharmaceuticalcomposition that is effective for treating one or more causes, symptomsor complications of PIH that occur or persist after delivery of thefetus, and particularly for treating symptoms or complications of PIHthat are not managed by or responsive to traditional antihypertensivedrugs or anti-convulsant or other agents used to manage seizures, HELLP,ocular or neurological deficits or disturbances, or any other symptom orcomplication of PIH that develops or continues postpartum.

Because the specific etiologies of gestational hypertension,preeclampsia, eclampsia and intrauterine growth restriction remainelusive, the medical definitions or diagnostic indicators of theseconditions continue to be revised from time to time. Thus, anyconventional diagnostic or prognostic method for assessment of the riskof or determining the presence of chronic or essential hypertension,gestational hypertension, preeclampsia, eclampsia or IUGR may be used inconnection with the invention. The inventions described herein are notlimited in any manner by the descriptions, definitions, diagnostic orclinical indications of PIH or IUGR described herein, and are deemed toinclude all existing and future revisions to the medical definitions ordiagnostic or prognostic indicators of gestational hypertension,preeclampsia, eclampsia, any other form of hypertension exhibited duringpregnancy, and intrauterine growth restriction.

BRIEF SUMMARY OF THE INVENTION

Pregnant women experiencing PIH or IUGR have serum levels of one or moreendogenous factors that are detectable by immunoassay for digoxin—acomponent of digitalis that is only found in certain plants. Theendogenous factors are present even though these pregnant women have notbeen poisoned or treated with digitalis. Because these endogenousfactors are not digoxin, anti-digoxin antibody treatment for digoxinpoisoning is not indicated for pregnant women. Furthermore, even if theendogenous factors in pregnant women were digoxin, pregnant women do notexhibit levels that warrant digoxin antibody treatment. The endogenousfactors in pregnant women, as measured by digoxin immunoassay, aredetected in the range of ≦0.1 ng/mL to 1.5 ng/mL. These endogenousfactor levels are substantially lower than the serum digoxin levelswhich are classified as life-threatening digoxin toxicity (i.e., greaterthan 6 ng/mL). Thus, one of ordinary skill in the art would not treatpregnant women with digoxin antibodies to counteract these low levels ofendogenous factors.

However, Applicant has surprisingly discovered that symptoms of PIH andIUGR may be effectively treated with high dose digoxin antibodycompositions. When presented herein in quotation marks, the term“digoxin” (e.g., serum “digoxin” concentration) means one or moreendogenous factor(s) that are detectable by an immunoassay for digoxin.Digoxin antibody compositions that are effective to treat PIH and IUGRhave digoxin binding capacity that is greater than the digoxin bindingcapacity that would be used to treat the serum “digoxin” concentrationsobserved in pregnancy if the endogenous factors were, in fact, digoxinand if comparable digoxin concentrations were deemed to be so poisonousor toxic as to justify digoxin antibody treatment.

Accordingly, the present invention relates to a pharmaceuticalcomposition for preventing, limiting the effects of, delaying the onsetof, or treating one or more of the causes, symptoms or complications ofgestational hypertension, preeclampsia, eclampsia or intrauterine growthrestriction. The invention also relates to a method of preventing,limiting the effects of, delaying the onset of, or treating gestationalhypertension, preeclampsia, eclampsia or intrauterine growth restrictionby administrating to a mammal a pharmaceutical composition having a highdose of digoxin binding capacity.

In one aspect of the invention, the pharmaceutical composition comprisesan antibody that reacts immunologically with or otherwise binds digoxinor digitoxin, having an active ingredient comprising a level of digoxinbinding capacity that is greater than the digoxin binding capacity thatwould be administered to neutralize digoxin or digitoxin toxicity in asubject having substantially the same serum digoxin concentration as theserum “digoxin” concentration observed in pregnant women (i.e., a highdose digoxin antibody composition).

The invention further relates to a pharmaceutical composition having atherapeutically effective amount of digoxin binding capacity (the activeingredient) sufficient to treat at least one cause, symptom orcomplication of PIH or IUGR. In one aspect of the invention, thecomposition has a therapeutically effective amount of the activeingredient to reduce systolic blood pressure, diastolic blood pressureor mean arterial pressure. In another aspect of the invention, thecomposition comprises a therapeutically effective amount of the activeingredient sufficient to cause a decrease in proteinuria. The inventionalso relates to a composition having a therapeutically effective amountof the active ingredient sufficient to cause an increase in urinaryoutput, an increase in creatinine clearance, or a reduction in serumcreatinine concentration. The invention also relates to a compositionhaving a therapeutically effective amount of digoxin binding capacitysufficient to cause a decrease in peripheral, cerebral or pulmonaryedema. The invention also comprises a composition having atherapeutically effective amount of digoxin binding capacity sufficientto cause an improvement in one or more neurological parameters,including, without limitation, clonus, hyper reflexia, central nervoussystem irritability, headache, eclamptic seizure, ocular function (e.g.,scotomata, double vision (diplopia), blurred vision, photophobia retinaldetachment, amaurosis and blindness), and fetal peri ventricular orintraventricular hemorrhage.

The invention also relates to a composition having a therapeuticallyeffective amount of digoxin binding capacity sufficient to cause anincrease in blood flow through a vein or artery, preferably a middlecerebral artery flow (maternal or fetal) or an umbilical artery orumbilical vein. The invention also relates to a therapeuticallyeffective digoxin antibody composition sufficient to increase nutrientdelivery, oxygenation, metabolic waste removal or fluid exchange betweenthe maternal circulation and the placenta and/or a fetus at risk fordeveloping or exhibiting intrauterine growth restriction.

The invention also comprises a composition having a therapeuticallyeffective amount of digoxin binding capacity sufficient to reduce orreverse inhibition of the sodium pump, or increase sodium pump activity,in the cells of a mammal exhibiting gestational hypertension,preeclampsia, eclampsia or intrauterine growth restriction.

The invention further relates to a composition comprising atherapeutically effective amount of digoxin binding capacity sufficientto cause a clinically beneficial reduction in systolic or diastolicblood pressure, mean arterial pressure, creatinine clearance, urinaryoutput, serum creatinine levels, or proteinuria. The invention furtherrelates to a composition comprising a therapeutically effective amountof digoxin binding capacity sufficient to cause a statisticallysignificant reduction in systolic or diastolic blood pressures, meanarterial pressure, serum creatinine level or proteinuria, or astatistically significant improvement in urinary output or creatinineclearance.

The invention also relates to a method of preventing, limiting theeffects of, delaying the onset of, or treating gestational hypertension,preeclampsia, eclampsia or intrauterine growth restriction, includingthe step of administering to a patient at risk for developing orsuffering from one or more symptoms of PIH or IUGR, a composition ofantibody that reacts immunologically with or otherwise binds digoxin,the composition having as the active ingredient digoxin binding capacitygreater than that which would be given to neutralize digoxinintoxication in a mammal having substantially the same serum digoxinconcentration as the serum “digoxin” concentration observed in pregnantwomen. The method may further include the step of administering multiplecompositions having different therapeutically effective amounts orrepeated administration of a composition having the same or a differenttherapeutically effective amount of the active ingredient.

These aspects are merely illustrative of the innumerable aspectsassociated with the present invention and should not be deemed aslimiting in any manner. These and other aspects, features and advantagesof the present invention will become apparent from the followingdetailed description when taken in conjunction with the drawings.Although methods and materials similar or equivalent to those describedherein may be used in the practice of the present invention, suitablemethods and materials are described below. In addition, the materials,methods and examples are illustrative only and not intended to belimiting in any manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating the steps and sequence of a methodfor treating gestational hypertension, preeclampsia, eclampsia orintrauterine growth restriction.

FIG. 2 is a diagram illustrating Doppler ultrasound wave shifts in anumbilical artery of a normotensive patient.

FIG. 3 is a diagram illustrating Doppler ultrasound wave shifts in anumbilical artery of a preeclamptic patient.

FIG. 4 a is a Doppler ultrasound chart illustrating blood flow velocitythrough a preeclamptic patient's umbilical artery before administrationof a high dose digoxin antibody composition.

FIG. 4 b is a Doppler ultrasound chart illustrating blood flow velocitythrough a preeclamptic patient's umbilical artery after administrationof a high dose digoxin antibody composition.

FIG. 5 a is an example of the aglycone structure of a cardenolide.

FIG. 5 b is an example of the aglycone structure of a bufadienolide.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Abbreviations

The following terms as used herein shall have the definitions set forthbelow. When such terms are used in the context as defined below, thensuch terms may be used in the abbreviated form set forth adjacent tosuch term.

“pregnancy-induced hypertension” or “PIH” mean and include one or moreof non-proteinuric gestational hypertension, preeclampsia and eclampsia,and include such conditions when occurring antepartum (whether before orafter 20 weeks' gestation) or postpartum. PIH may be superimposed overother forms of hypertension, such as chronic or essential or secondaryhypertension. Accordingly, for purposes of the invention PIH includesany form of hypertension when present during pregnancy. When PIH issuperimposed over existing forms of hypertension the prognoses formother and fetus are much worse than either condition alone.Superimposed PIH is the likely diagnosis (1) where new onset proteinuriaoccurs when non-proteinuric hypertension is present early in pregnancy(before 20 weeks' gestation), and (2) in women with hypertension andproteinuria before 20 weeks' gestation if there is thrombocytopenia(platelet count≧50,000/mm³ and ≦100,000 cells/mm³), increase of alanineor aspartate aminotransferase or alkaline phosphatase (together, “liverenzymes”) to abnormal levels, sudden increase in proteinuria, or suddenincrease in blood pressure where hypertension has previously been wellcontrolled. Furthermore, any pregnant woman whose blood pressureincreases by 30 min Hg systolic or 15 mm Hg diastolic (even if it doesnot exceed 140 mm Hg systolic or 90 mm Hg diastolic) may be consideredto have PIH. Additional symptoms and complications of PIH may include:peripheral edema; exaggerated myocardial function, vasoconstriction andvasospasm; eclamptic seizures; hemolysis, elevated liver enzyme levelsand low platelets (when occurring together, referred to as HELLPsyndrome); hepatic rupture, elevated blood urea nitrogen (“BUN”) orbilirubin or lactate dehydrogenase (“LDH”); pulmonary edema; acute renalfailure; placental abruption; IUGR or intrauterine fetal demise;cerebral edema and cerebral hemorrhage (including fetal periventricularand intraventricular hemorrhage); scotomata, double or blurred vision,amaurosis, cortical blindness, retinal detachment; and neurologicaldisturbances (headache, altered consciousness or mental status, hyperreflexia, central nervous system irritability, clonus).

“proteinuria” means the urinary excretion of at least 0.3 gm protein ina 24-hour specimen, which usually correlates with at least 30 mg/dL (≧1+on dipstick), with no evidence of urinary tract infection.

“gestational hypertension” means non-proteinuric hypertension duringpregnancy. Gestational hypertension is generally characterized as theacute onset of hypertension in pregnancy or the early puerperium withoutproteinuria. Gestational hypertension usually resolves within ten (10)days after delivery. Currently, a diagnosis of gestational hypertensionrequires a blood pressure of at least 140 mm Hg systolic or 90 mm Hgdiastolic measured at least two times and at least six hours apart, thatdevelops after 20 weeks' gestation in a previously normotensive patient.Sustained blood pressure measurements over 160 mm Hg systolic or 110 mmHg diastolic are classified as severe gestational hypertension.

“preeclampsia” means the occurrence of high blood pressure andproteinuria during pregnancy (usually after 20 weeks' gestation).Preeclampsia is further characterized as either mild or severe.Currently, the American College of Obstetricians and Gynecologists(“ACOG”) defines mild preeclampsia as the occurrence in pregnant womenof hypertension greater that 140 mm Hg systolic or 90 mm Hg diastolicand proteinuria greater than 300 mg per 24 hours. ACOG currently definessevere preeclampsia as preeclampsia with one or more of the following:(1) hypertension greater than 160 mm Hg systolic or 110 mm Hg diastolic,as measured on two occasions at least six hours apart while on bed rest,(2) a proteinuria level of greater than 500 mg per 24 hour period or 3+or greater on two random urine samples collected at least 4 hours apart,(3) thrombocytopenia (platelets≧50,000/mm³ and ≦100,000/mm³) ormicroangiopathic hemolysis, (4) elevations in alanine aminotransferase(ALT(SGPT)>72 U/L) or aspartate aminotransferase (AST(SGOT)>72 U/L),lactate dehydrogenase (LDH>600 U/L) or total bilirubin (>1.2 mg/dL),epigastric or right upper quadrant pain, (5) persistent headache orvisual scotomata or other visual disturbances, (6) intrauterine growthrestriction, (7) oliguria (urinary output less than or equal to 500 mLper 24 hours or average of ≦25 ml/hour over a 3 hour period), serumcreatinine level of less than 1.2 mg/dL, or (8) pulmonary edema.Although not specifically included in the ACOG definition, generalswelling or peripheral edema, especially when it occurs by rapid onset,may also indicate preeclampsia. Peripheral edema may be determined on ascale of 0 to 4+, with 0 representing no edema, 1+ barely detectable“pitting” or tissue indentation, 2+ “pitting” of <5 mm, 3+ “pitting” of5-10 mm, and 4+ “pitting” of >10 mm. Peripheral edema is frequentlyevaluated in the leg, sacrum, hand, and face.

“eclampsia” means mild or severe preeclampsia together with seizures.Eclampsia may be further characterized by diminished or alteredneurological function, mental status, severe agitation, confusion andunconsciousness for a variable period of time.

“intrauterine growth restriction” or “IUGR” mean intrauterine growthrestriction or growth retardation of a fetus. IUGR is currentlycharacterized by a fetus whose estimated weight is below the 10^(th)percentile for its gestational age and a maternal abdominalcircumference below the 2.5 percentile for gestational age. IUGR mayresult in or contribute to intrauterine fetal demise. For purposes ofthe invention, to prevent, limit the effects of, or delay the onset ofIUGR includes to prevent, limit the effects of, or to delay intrauterinefetal demise.

“sodium pump” or “sodium/potassium ATPase” or “Na⁺/K⁺ ATPase” mean thetransmembrane protein that utilizes energy generated from adenosinetriphosphate (“ATP”) hydrolysis (ATP→ADP+PO₄) to transport sodium andpotassium ions across cell membranes in opposite directions againsttheir chemical and electrical gradients. The sodium pump is the primarytransporter responsible for maintaining the electrochemical gradient ofNa⁺ across cell membranes and is important in regulating cell volume,cytoplasmic pH, Na⁺-dependent glucose and amino acid transport, andregulating Ca²⁺ levels through the Na⁺/H⁺ and Na⁺/Ca²⁺ ion exchangepumps. The sodium pump is regulated by multiple mechanisms in responseto changes in cellular requirements or cellular environments.

“endogenous factors” or “EFs” mean those factors produced in a mammalthat cross-react immunologically with antibodies to at least onecardenolide or bufadienolide. Sometimes herein, EFs are also referred toas digoxin-like substances (“DLS”), digoxin-like immunoreactivesubstances or factors (“DLIS” or “DLIF”), or endogenous digoxin-likefactors (“EDLF”).

“cardenolides (and/or) bufadienolides” or “cardiac glycosides” meancardenolides and/or bufadienolides, and aglycones thereof, including,without limitation, digitalis, gitoxigenin, digoxigenin, digoxin,digitoxigenin, digitoxin, dihydrodigoxin, strophanthins, convallatoxin,cymarine, acetylstophanthidin, strophanthidin, ouabagenin, ouabain,dihydrooubain, neriifolin, proscillaridin, proscillaridin A,cinobufagen, cinobufatolin, marinobufagenin, norbufalin, bufanolide,bufalin and similar compounds, and their respective isomers, inotropes,congeners, variants, derivatives, equivalents, precursors andmetabolites, and synthetic versions of any of the foregoing.

“therapeutically effective” means effective to treat a disease, syndromeor condition. As used herein, “treat” includes, without limitation, toprevent (prophylactic), reduce the severity of, control, limit theeffects of, delay the onset of, alleviate or ameliorate one or more ofthe causes, symptoms, indications or complications of a disease,syndrome or condition. A therapeutically effective composition does notcause any unacceptable or significant adverse effect in the subject,including the worsening of any condition, complication or symptom tosuch an extent that the risks of the adverse effect outweigh thebenefits to be derived from the treatment. A therapeutically effectivecomposition may improve or stabilize one or more medical parametersthat, in the absence of the composition, might otherwise worsen ordevelop into a symptom or complication of a disease, condition orsyndrome.

“digoxin antibody” or “digoxin antibodies” mean an antibody or bindingfragment that reacts immunologically with or binds to (eitherspecifically or non-specifically) or contains a binding domain fordigoxin, digitoxin, digitoxigenin, digoxigenin, or gitoxingenin. Digoxinantibodies may be specific to and include antibodies against anycardenolide or bufadienolide that has the ability to bind at least oneepitope of digoxin or digitoxin, digitoxigenin, digoxigenin, orgitoxingenin. Digoxin antibodies include, without limitation, antibodiesspecific to digoxin, ouabain, bufalin, and marinobufagenin, or to aconjugate of any one of the foregoing. Digoxin antibodies also includecalycin proteins, whether naturally occurring or wholly or partiallysynthetic or engineered, that are capable of binding digoxin, digitoxin,digitoxigenin, digoxigenin, or gitoxingenin. The calycin superfamily ofproteins are characterized by structural motifs formed by anti-parallel,beta-sheets in a manner similar to the CDR region of immunoglobulins.Lipocalins, fatty acid-binding proteins (“FABPs”) and avidins aremembers of the calycin superfamily of proteins. Calycins are relativelysmall secreted proteins that are believed to be involved in the bindingand transport of hydrophobic molecules. The specificity of binding isdetermined by the conformation and constituent side-chains of the pocketcreated by folding of the protein. In vitro, many lipocalins can bindwith high affinity to a range of hydrophobic molecules not normallyencountered in nature. This may represent an inherent ability of theproteins to bind molecules having particular biochemical and structuralproperties. A calycin can be functionally divided into a “bindingdomain” and a “targeting domain.” The “binding domain” functions tointeract with ligands, while the “targeting domain” functions to providespecificity in transporting the bound ligand to a defined site. Knownlipocalins include: retinol-binding protein; purpurin; retinoicacid-binding protein; alpha.sub.2u-globin; major urinary protein;bilin-binding protein; alpha-crustacyanin; pregnancy protein 14;beta.-lactoglobin; neutrophil lipocalin and choroid plexus protein;odorant-binding protein; von Ebner's gland protein; probasin; andaphrodisin. Lipocalins appear to have a regulatory influence on theinflammatory cascade and protect against excessive tissue damage. See,Flower D, FEBS Letters 354:7-11 (1994); Flower D, J. Molec. Recognition8:185-195 (1995); Fowler D, Biochem. J. 318:1-14 (1996). Calycinproteins may be engineered to have a prescribed ligand specificity, asdescribed in Schlehuber S and Skerra A, Biophysical Chemistry 96:213-228(2002), U.S. Patent Application 20050106660 and PCT Applications WO99/16873, WO 00/75308 (each of which applications is incorporatedherein). As described in the foregoing PCT applications, thebilin-binding protein lipocalin has been specifically engineered to binddigoxigenin and therefore is included within the definition of digoxinantibody.

“digoxin binding capacity” means the amount of digoxin that is capableof being bound by a given amount of digoxin antibody. For example, asingle vial of DIGIBIND® contains 38 mg total digoxin antibody and iscapable of binding approximately 0.5 mg digoxin, i.e., one vial has 0.5mg digoxin binding capacity. Thus, a vial having this formulation ofDIGIBIND® has digoxin binding capacity of 0.013 mg digoxin or digitoxinbound per mg of antibody (0.5 mg digoxin÷38 mg total antibody).Similarly, a vial of the current formulation of DIGIFAB™ has 0.0125 mgdigoxin or digitoxin bound per mg of antibody (0.5 mg÷40 mg totalantibody).

“high dose” means an amount of a digoxin antibody composition havingdigoxin binding capacity that is greater than the digoxin bindingcapacity that would be used to treat the serum “digoxin” concentrationsobserved in pregnancy if the endogenous factors were, in fact, digoxinand if comparable digoxin concentrations were deemed to be poisonous ortoxic so as to warrant treatment with digoxin antibodies. A high dose isan amount of digoxin antibody having digoxin binding capacity that isgreater than the digoxin binding capacity in a single application dosefor neutralizing digoxin/digitoxin toxicity in a subject havingsubstantially the same serum digoxin/digitoxin concentration as theserum “digoxin” concentration observed in a pregnant woman. Preferably,when administered to treat a condition in which clinical symptoms of PIHor IUGR are present, a high dose comprises digoxin binding capacity ofmore than 0.006 mg per Kg patient weight; however, when administered totreat PIH or IUGR before manifestation of clinical symptoms (i.e., forprophylaxis), a high dose composition may also comprise digoxin bindingcapacity of ≦0.006 mg per Kg patient weight. More preferably, thedigoxin binding capacity of a high dose digoxin antibody composition isbetween three-fold and one hundred-fold, and most preferably ten-fold,the digoxin binding capacity that would be used for treatingdigoxin/digitoxin intoxication in a patient having substantially thesame serum digoxin/digitoxin concentration as the serum “digoxin”concentration observed in a pregnant woman. The high dose compositionmay be selected based upon either quantified serum “digoxin” or“digitoxin” concentration (such as by immunoassay) or based upon theamount believed to be present in the subject based upon the range ofserum “digoxin” or “digitoxin” concentrations generally known to bepresent in pregnant patients, whether or not experiencing one or moresymptoms of PIH or IUGR, and/or based upon the severity of PIH or IUGRsymptoms or the risk factors (in kind or in number) for a patient todevelop PIH or IUGR.

“antibody” or “antibodies” mean an immunoglobulin or any polypeptide orprotein, whether natural or partly or wholly synthetically engineered orproduced, having a binding domain which is, or is substantiallyhomologous to, a binding domain for an antigen or an epitope of anantigen. Examples of antibodies are the immunoglobulin isotypes andtheir isotypic subclasses; antigen binding fragments; and diabodies (asdefined below). The term also includes derivatives, functionalequivalents and homologies of antibodies, including any polypeptidecomprising a binding domain, whether natural or wholly or partiallysynthetic, polyclonal, monoclonal, humanized, chimeric or fully human.Various methods of producing such antibodies are described in U.S. Pat.No. 5,225,539, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,869,619, U.S.Pat. No. 5,821,337, U.S. Pat. No. 5,859,204, U.S. Pat. No. 6,946,546,U.S. Pat. No. 6,939,543, U.S. Pat. No. 6,926,896, U.S. Pat. No.6,924,125, U.S. Pat. No. 6,893,624 and U.S. Pat. No. 6,881,557, each ofwhich patents is incorporated herein by this reference.

“antigen binding fragments” or “binding fragments” mean fragments ofwhole antibodies and naturally occurring or synthetically constructedmolecules that have a binding domain, whether specific or non-specific,for a ligand, an antigen or an epitope of an antigen.

“diabodies” mean multimers of polypeptides, each polypeptide comprisinga first domain comprising a binding region of an immunoglobulin lightchain and a second domain comprising a binding region of animmunoglobulin heavy chain, the two domains being linked (e.g. by apeptide linker) but unable to associate with each other to form abinding domain. Binding domains are formed by the association of thefirst domain of one polypeptide within the multimer with the seconddomain of another polypeptide within the multimer (US Patent App.2005/0214860, incorporated herein by this reference).

“binding domain” means the part of an antibody or of any other naturallyoccurring or synthetically engineered or constructed molecule,including, without limitation, a calycin protein, that binds to and/oris complementary to a part of or all of a ligand or an antigen or anepitope of an antigen. For purposes of the invention, an endogenousfactor is deemed to be a ligand or an antigen.

“complementarity determining region” or “CDR” mean one or more of thethree hypervariable sequences in the variable regions within each of theimmunoglobulin light and heavy chains.

“mean arterial pressure” or “MAP” mean the pressure determined by eitherof the following formulae: [(2×diastolic pressure)+systolic pressure]÷3OR diastolic pressure+⅓ (systolic pressure−diastolic pressure)

“S/D ratio” means, with respect to Doppler ultrasound, the ratio betweenthe Doppler frequency shift during systolic flow and end diastolic flowin an artery or vein.

“RI” means, with respect to Doppler ultrasound, the resistance index ofan artery or vein as determined by the formula (S−D)÷S, where “S”represents the Doppler frequency shift during systolic flow and “D”represents the Doppler frequency shift during end diastolic flow.

“creatinine clearance” or “CrCl” mean the test comparing the level ofcreatinine in urine with the creatinine level in the blood, usuallybased on measurements of a 24-hour urine sample and a blood sample drawnat the end of the 24-hour period. Clearance is often measured asmilliliters/minute (mL/min). Creatinine clearance is used to estimatethe glomerular filtration rate (“GFR”). The GFR, in turn, is thestandard by which kidney function is determined. CrCl for women may bedetermined using the formula:CrCl=(140−age)×(weight)×0.85/(sCr×72) (“sCr”=serum creatinineconcentration).

“mg” means milligram or milligrams.

“μg” or “mcg” means microgram or micrograms.

“ng” means nanogram or nanograms

“Kg” means kilogram or kilograms.

“mL” means milliliter or milliliters.

“Apgar” means the neonatal health assessment made after birth,preferably at the first and fifth minutes after birth, for each of:activity and muscle tone; pulse (heart rate); grimace (reflexirritability); appearance of skin coloration; and respiration (rate andeffort). Each activity receives a score of 0 (lowest), 1 or 2 (highest).A total score of 7 or above, either on the first minute testing or asimproved at the fifth minute testing, is generally considered toindicate good health.

“biophysical profile score” or “biophysical profile” or “BPS” mean theassessment of fetal wellness as determined by evaluation (biophysicalprofile procedure or “BPP”) over a period of thirty minutes of (1) thenon-stress test (“NST”), (2) ultrasound measurement of amniotic fluidvolume (“AFV”), (3) presence/absence of fetal breathing movements, (4)gross body movements, and (5) tone. Each parameter receives a score ofnormal=2, abnormal=0 (for a maximum total score of 10 points).Alternatively, a modified BPP may initially evaluate only NST and AFVassessments and if either is abnormal then a complete BPP is performed.

BIOPHYSICAL PROFILE SCORE - INTERPRETATION & TREATMENT Risk of FetalRisk (%) of Death Result Interpretation Asphyxia* (per 1000 per wk)Recommended Treatment 10/10 Non-asphyxiated 0 0.565 Conservative 8/10normal AFV Non-asphyxiated 0 0.565 Conservative 8/8 w/o NSTNon-asphyxiated 0 0.565 Conservative 8/10 ↓ AFV Chronic 5-10 20-30 If≧37 wk - deliver compensated If <37 wk - serial testing asphyxia 6/10normal AFV Acute asphyxia 0 50 If ≧37 wk - deliver possible If <37 wk -repeat test w/in 24 hr. and if <6/10 - deliver 6/10 ↓ AFVChronic >10 >50 Factor in gestational age asphyxia w/ If ≧32 wk -deliver possible acute If <32 wk - test daily 4/10 normal AFV Acuteasphyxia 36 115 Factor in gestational age likely If ≧32 wk - deliver If<32 wk - test daily 4/10 ↓ AFV Chronic >36 >115 If ≧26 wk - deliverasphyxia w/ acute asphyxia likely 2/10 normal AFV Acute asphyxia 73 220If ≧26 wk - deliver almost certain 0/10 Gross severe 100 100 If ≧26 wk -deliver asphyxia *Umbilical venous blood pH less than 7.25

“Clinical Global Impression” or “CGI” mean the clinical assessment ofseverity of a patient's condition at the time of evaluation (“CGI-S”),and the improvement in a patient's condition since a prior evaluation orinitial severity screen (“CGI-I”). Maternal factors to assess CGIinclude, without limitation: physical examination, mental status, ECG,clinical laboratory results, vital signs, patient/family member reports,and medical professional reports. Fetal factors to assess for CGIinclude, without limitation: heart tracing, biophysical profile, andmedical professional reports. The CGI is designed to document overallclinical impression of the patient's overall condition. The ClinicalGlobal Impressions Scale described below has been modified from thescale presented by Guy by limiting the potential ratings to four for theSeverity of Illness Scale and to three for the Improvement Scale. Theoriginal CGI-S scale is as follows: 1. Normal, not ill at all; 2.Borderline ill; 3. Mildly ill; 4. Moderately ill; 5. Markedly ill; 6.Severely ill; 7. Among the most extremely ill patients. The originalCGI-I is as follows: 1. Very much improved; 2. Much improved; 3.Minimally improved; 4. No change; 5. Minimally worse; 6. Much worse; and7. Very much worse. See, Guy W., ECDEU Assessment Manual forPsychopharmacology—Revised, Rockville, Md., U.S. Dept. Health and HumanServices [Publ No ADM 76-338] (1976, pgs. 218-222). Although the CGI-Sand CGI-I scales described below are preferred, for purposes of theinvention the original Guy scales or any modified form of a CGI-S and/orCGI-I scale may be used.

Improvement Severity Scale (CGI-S) (or Change) Scale (CGI-I) 1. Normal,not ill at all. 1. Improved. 2. Mildly ill. 2. No change. 3. Moderatelyill. 3. Worse. 4. Severely ill.

II. Etiology of PIH and IUGR

In the early stages of pregnancy, the placenta has a “loose” connectionwith the uterine wall. Around the fourteenth to sixteenth week of normalpregnancy, the placenta develops a “tight” attachment to the uterinewall in order to tap more fully into the maternal blood supply. Duringthis normal placental development, trophoblasts invade the maternalspiral arterioles and completely remodel the maternal spiral arteriolesinto large capacitance vessels with low resistance, allowing for theexchange of larger and more complex nutrients that are required for thecontinued development of the fetus at the later stages of the pregnancy.Furthermore, during normal differentiation, in a process known aspseudovasculogenesis, invasive trophoblasts alter their adhesionmolecule expression from those characteristic of epithelial cells(integrin α₆/β₃ and αω/β₅, and E-cadherein) to those of endothelialcells (integrin α₁/β₁ and αω/β₃, platelet endothelial cell adhesionmolecule and vascular endothelial-cadherin). In preeclampsia, shallowplacental trophoblast invasion of the uterine spiral arterioles leads toreduced placental perfusion and placental insufficiency.

PIH is known to cause vasoconstriction and vasospasm, damaging thesmooth muscle lining of the blood vessels. This damage to blood vesselscan also lead to edema, including cerebral edema. Vasospasm can occurthroughout the body, damaging the heart, kidneys, liver and brain.Vascular damage leads to the accumulation of platelets in the bloodvessels, forming small clots along the blood vessel wall and furthernarrowing the blood vessel, resulting in further vasoconstriction. Thiscascade of events exacerbates the severity of the maternal hypertensivecondition and increases the risk for developing or the severity of IUGR.

The fetus is connected by the umbilical cord to the placenta andreceives nutrition and oxygenation from the maternal circulation throughthe placenta. Oxygenated blood travels from the placenta through theumbilical vein to the fetal heart, where it then is distributed to fetaltissues and is finally returned (along with metabolic wastes) to theplacenta through two umbilical arteries. In PIH, hypertension,vasoconstriction, vasospasm and exaggerated myocardial function may,individually or together, negatively impact the flow of blood to theplacenta, and consequently to the fetus, such that little or no bloodflows to the placenta and fetus (i.e., there is insufficient placentaland fetal perfusion).

FIG. 2 illustrates the blood flow velocity in the umbilical artery of anormal pregnancy. In FIG. 2, “A” refers to the blood flow velocityduring systole, while “B” refers to the blood flow velocity in theumbilical artery during end diastole. FIG. 2 illustrates that in anormal pregnancy, even during diastole, there is a significant flowvelocity through the umbilical artery. This is in contrast to the flowvelocity in a preeclamptic patient, as illustrated in FIG. 3. In PIH,reduced fetal perfusion is evidenced by umbilical artery flow velocity“bottoming out” (being reduced or absent) during diastole, indicated inFIG. 3 by “C”. In the most severe circumstance, the direction ofumbilical artery flow may become reversed. Maternal vasoconstrictionresults in restricted blood flow to and through the fetus and reducedflow (and disposal of metabolic wastes) from the fetus through theumbilical artery back to the maternal circulation. Reduced placental andfetal perfusion results in reduced oxygenation and fluid exchange(nutrition and waste removal) and may cause or contribute to IUGR.

The reduced placental perfusion makes the exchange of nutrients andmetabolic waste removal through the placenta more difficult. It is knownthat many nutrient transport processes across the placenta (specificallyacross the microvillus membrane of syncytiotrophoblasts) aresodium-coupled and are dependent upon the maintenance of a lowintracellular Na⁺ concentration. It is also known that sodium/potassiumATPase activity is reduced in the microvillus membrane ofsyncytiotrophoblasts isolated from the placentas of IUGR pregnancies.Applicant theorizes that inhibition of sodium/potassium ATPase may causeor contribute to IUGR because of the reduced ability of the placenta andfetus to receive nutrients and dispose of metabolic wastes necessary forcontinued fetal growth and development.

III. Endogenous Sodium Pump Inhibitors in PIH and IUGR

A. Endogenous Factors Associated with PIH and IUGR.

It is theorized by Applicant that the failure of the placenta toproperly invade the uterine wall is associated with the release ofabnormal levels of endogenous factors from placental, fetal and/ormaternal sources. Endogenous sodium pump inhibitors are found inpatients having mineralocorticoid hypertension (primary aldosteronismand ectopic corticotrophin syndrome), essential hypertension andhypertension in plasma-volume expanded states, including normotensivepregnancy and pregnancy complicated by PIH. It is known that theseendogenous factors inhibit Na⁺/K⁺ ATPase activity in vitro. Applicanthas discovered that, in vivo, red blood cells of preeclamptic patientsexhibit decreased sodium pump activity and that this sodium pumpinhibition is reduced or reversed by administration of high dose digoxinantibody compositions to the preeclamptic patient. Thus, it has beendiscovered by Applicant that these endogenous factors cause orcontribute to one or more symptoms or complications of PIH or IUGR.

These endogenous factors have not been fully characterized but have beengenerally referred to as digitalis-like or digoxin-like because theyhave physical, chemical and biological characteristics similar to thoseof cardenolides and bufadienolides, which are known cardiotonicsteroids. The endogenous factors have been variously described as“endoxin,” “endobain,” “digoxin-like,” “digitalis-like,” dihydrodigoxin,“endogenous ouabain,” “ouabain-like,” dihyrooubain, “proscillaridinA-like,” “endogenous marinobufagenin,” “marinobufagenin-like,”“bufalin-like” and 19-norbufalin. Digitalis-like, ouabain-like,bufalin-like and marinobufagenin-like endogenous factors have beenspecifically observed in neonates, normotensive pregnancy and in PIH.

It is believed by Applicant that these EFs may inhibit sodium pumpactivity through direct or indirect binding of one or more isoforms ofthe sodium pump or of other membrane-bound proteins that influencesodium pump activity, or through regulation of sodium pump expression,degradation or recycling, or through some other regulatory mechanismdirectly or indirectly affecting the sodium pump.

B. Cardiac Glycosides—Exogenous Sodium Pump Inhibitors.

Cardiac glycosides are composed of two structural features, the sugar(glycoside) and the non-sugar (aglycone) steroid moieties. The glycosidemoiety may not significantly affect sodium pump binding affinity but mayalter the biopharmaceutical or pharmacokinetic properties of the cardiacglycoside. See, Pullen, M. A., et al., J. Pharm. Exp. Therapeutics310(10): 319-325 (2004). Aglycones of the general class of cardenolidesand bufadienolides are shown, respectively, in FIGS. 5 a and 5 b.

Cardenolides and bufadienolides are known to specifically inhibit Na⁺/K⁺ATPase. The sodium pump is responsible for establishing and maintainingthe electrochemical gradient of sodium and potassium ions across theplasma membrane of mammalian cells, maintaining the intracellularcytoplasm high in K⁺ and low in Na⁺. The presence of a binding site forexogenous sodium pump inhibitors suggests that one or more endogenous“digoxin-like” factors may be involved in the regulation of sodiumhomeostasis, blood pressure and volume.

C. Antibodies to Cardiac Glycosides as Therapeutic Agents.

Digitalis or its constituents, digoxin and digitoxin, are the primarycardiotonic steroids that are used therapeutically to treat cardiacarrhythmias and congestive heart failure. Digoxin and digitoxin have anarrow therapeutic range (1.0-1.9 nmol/L or approximately 0.8-1.5 ng/mLserum digoxin concentration) and overdose to these drugs is notuncommon. Digoxin overdose and life-threatening digoxin toxicity aretreated through the administration of antibodies to digoxin. It isbelieved that antibodies counteract the effects of digoxin or digitalisbecause the binding domain of the antibody binds to the cardiacglycoside thereby preventing it from binding to or otherwise inhibitingor regulating the expression or function of Na⁺/K⁺ ATPase. If theantibodies are specific to the cardiac glycoside they will bind it withhigh affinity, favoring movement of the cardiac glycoside out of tissueand allowing the resulting antigen/antibody complex to be eliminatedfrom the body. Reversal of Na⁺/K⁺ ATPase inhibition caused by cardiacglycosides is believed to be due to the greater affinity of theantibodies for the cardenolide or bufadienolide, than the affinity ofthe cardenolide or bufadienolide for Na⁺/K⁺ ATPase.

D. Role of Endogenous Sodium Pump Inhibitors in PIH and IUGR.

Applicant has theorized that elevated levels of EFs in PIH and IUGRinhibit the normal functioning of, or regulate at least some isoforms orsubunits of, Na⁺/K⁺ ATPase, causing intracellular levels of sodium andcalcium ions to rise. When normal vascular endothelial cell osmolalityis altered, an influx of water may rupture the endothelial cells causingan inflammatory response that further constricts the interior diameterof the vasculature, thereby contributing to the hypertensive state.Vasoconstriction may produce exaggerated myocardial function due to theincreased effort required by the heart to pump blood through thenarrowed vasculature. It is also possible that exaggerated myocardialfunction results from direct action of the endogenous factor(s) oncardiac muscle cells. Vascular responsiveness to vasoactive agents mayalso be affected by deviations from the normal balance of sodium,potassium and calcium ions within the vascular endothelia. It isbelieved by Applicant that this intracellular ion imbalance within cellsof the placenta, vasculature, brain and/or kidneys leads to, inter alia,intravascular volume contraction, vasoconstriction, vasospasm and maylead to or contribute to IUGR. Furthermore, alteration in the capacityof the syncytiotrophoblast Na⁺/K⁺ ATPase to maintain the Na⁺ gradientmay indirectly affect the sodium-dependent or sodium-coupled transportsystems of the placenta (such as amino acid and phosphate uptake),impairing nutrient and oxygen supply and extrusion of protons (H⁺) fromthe syncytiotrophoblasts, resulting in or contributing to IUGR.

It is known that in vitro sodium pump inhibition by EFs may be reversedor prevented by addition of antibodies to cardenolides andbufadienolides, particularly anti-digoxin antibodies. Pullen, M. A, etal., J Pharm. Exp. Therapeutics 310:319-325 (2004). Applicant hasdiscovered that in vivo administration of a high dose digoxin antibodycomposition also reduces or reverses sodium pump inhibition or otherwiseincreases sodium pump activity. Thus, it is believed that antibodies tocardenolides and bufadienolides may be useful in diagnosing, preventingand/or treating PIH.

VI. Therapeutic Antibodies in General

Passive immunity is conferred when antibodies generated in one sourceare administered to a different source in order to prevent or treat adisease or an infection, or to neutralize toxicity. Applicant hasdiscovered that antibodies used to treat cardiotonic steroidintoxication in humans are also effective for treating the causes orsymptoms of PIH and IUGR. Applicant believes that these antibodies bindto EFs such that the antibody-EFs complex may be removed from the body,effectively treating or controlling one or more of the causes or thesymptoms or complications of PIH and IUGR. If symptoms or complicationsor causes of PIH or IUGR may be treated or controlled, ultimately thepregnancy may be continued, premature delivery may be delayed oravoided, fetal development may continue and the maternal and fetalconditions and outcomes may be improved.

A. Introduction to the Immune System.

Blood is comprised of a variety of cells, including platelets, red bloodcells and leucocytes (monocytes, neutrophils, eosinophils, mast cells,basophils) and circulating lymphocytes (B-cells and T-cells). Eachleucocyte and lymphocyte has a different responsibility, but allfunction together for the primary objective of recognizing, attackingand destroying bacteria, viruses, cancer cells, and all substances seenas foreign.

The major function of B lymphocytes is the production of antibodies inresponse to foreign substances, including bacteria, viruses, and tumorcells. Antibodies are specialized proteins that specifically recognizeand bind to one particular molecule or protein. Each B lymphocyteexpresses a surface antibody having a unique binding domain for oneantigen. When an antigen is large, an antibody may only bind to aparticular part of the antigen, which part is referred to as an epitope.Antibody binding is generally specific for one antigen or an epitope ofan antigen, and the antigen-antibody binding is usually of highaffinity.

The unique binding domains of antibodies are not the result of exposureto the antigen, but arise through random genetic rearrangements. When aB-cell encounters an antigen, the B-cell is activated to differentiateinto a plasma cell, secreting different classes of antibodies (such asIgG, IgA or IgM) having the same binding domain. Antigen recognition andbinding allows antibodies to initiate the complement system, a complexbiochemical cascade of more than 35 serum protein that leads to, interalia, opsonization and cytolysis of antigen presenting cells (foreigncells or macrophages/neutrophils that have small portions of digestedantigens on the exterior surface of their cell membranes).

Opsonization is a process through which bacteria, virus-infected cellsand other cells are targeted for destruction. Antibodies serve as “tags”(opsonins) because one end (the Fab) binds to an antigen on the foreigncell while the other end of the antibody (the Fc) binds to receptors onphagocytic cells (e.g., macrophages and neutrophils). Theantibody-antigen complex signals the phagocyte to engulf and destroy theantigenic organism or cell. Activation of the complement system alsocauses formation of an aggregate of complement proteins (Membrane AttackComplex or “MAC”). MAC is capable of inserting itself into the cellmembrane of an antigen presenting cell, creating holes that allow ions,water and other small molecules to freely pass through the membrane (theMAC attack). As a result, the antigen presenting cell will not be ableto maintain its osmolality and will quickly die or undergo cytolysis.

B. Antibodies in General.

All naturally occurring whole antibodies have a common core structure oftwo identical light chains, each being about 24 kilodaltons, and twoidentical heavy chains each being about 55-70 kilodaltons. One lightchain is attached to each heavy chain, and the two heavy chains areattached to each other. Both the light and heavy chains contain a seriesof repeating homologous units, each of about 110 amino acid residues inlength which fold independently in a common motif called animmunoglobulin (Ig) domain. All Ig domains contain the complementaritydetermining regions (“CDR”), which are specific for and bind to theantigen or epitope. There are between 10⁸ and 10¹⁰ structurallydifferent antibody molecules in every individual. Antibody sequencediversity is predominantly found in three short amino acid sequenceswithin the amino terminal variable domains of the heavy and lightchains, called the hypervariable regions, to distinguish them from themore conserved “framework regions” that flank each CDR within thevariable regions of the light and heavy chains.

Despite their overall similarity, antibody molecules can be divided intodistinct classes and subclasses based on physiochemical characteristicssuch as size, charge and solubility, and on their behavior in binding toantigens. In humans, the classes of antibody molecules include IgA, IgD,IgE, IgG and IgM. Members of each class are said to be of the sameisotype. IgA and IgG isotypes are further subdivided into subtypescalled IgA.sub.1, IgA.sub.2 and IgG.sub.1, IgG.sub.2, IgG.sub.3 andIgG.sub.4. The heavy chains of all antibody molecules in an isotypeshare extensive regions of amino acid sequence identity, but differ fromantibodies belonging to other isotypes or subtypes. Heavy chains aredesignated to the overall isotype of the antibody molecule, e.g., IgAcontains “alpha”, IgD contains “delta”, IgE contains “epsilon”, IgGcontains “gamma”, and IgM contains “mu”. IgG, IgE and IgD circulate asmonomers. IgA circulates as a monomer, and molecules secreted throughthe epithelia into the mucosal lining of body cavities are homodimers.IgM molecules form pentamers.

C. Production of Antibodies.

Animals may be inoculated with an antigen in order to produce antibodiesspecific for the antigen. Frequently an antigen is bound or conjugatedto another molecule to enhance the immune response. As used herein, aconjugate is any peptide, polypeptide, protein or non-proteinaceouscompound bound to an antigen that is used to elicit an immune response.Antibodies produced in an animal in response to antigen inoculationcomprise a variety of non-identical molecules (polyclonal antibodies)made from a variety of individual antibody producing B lymphocytes. Theepitope is the specific antigenic structure recognized by the bindingdomain. A polyclonal antibody is a mixed population of antibody species,each of which may recognize a different epitope on the same antigen.Given the correct conditions for polyclonal antibody production in ananimal, most of the antibodies in the animal's serum will recognize thecollective epitopes on the antigenic compound to which the animal hasbeen immunized. This specificity is further enhanced by affinitypurification to select only those antibodies that recognize the antigenor epitope of interest.

A monoclonal antibody is a single species of antibody wherein everyantibody molecule recognizes the same epitope on the antigen because allantibody producing cells are derived from a single B-lymphocyte cellline. Hybridoma technology involves the fusion of a single B lymphocytewith an immortal myeloma cell (usually mouse myeloma). This technologyprovides a method to propagate a single antibody-producing cell for anindefinite number of generations, such that unlimited quantities ofstructurally identical antibodies having the same antigen specificity(monoclonal antibodies) may be produced. However, in therapeuticapplications a goal of hybridoma technology is to reduce the immunereaction in humans that may result from administration of monoclonalantibodies generated by the non-human (e.g. mouse) hybridoma cell line.

Methods have been developed to replace light and heavy chain constantdomains of the monoclonal antibody with analogous domains of humanorigin, leaving the variable regions of the foreign antibody intact.Alternatively, “fully human” monoclonal antibodies are produced in micetransgenic for human immunoglobulin genes. Methods have also beendeveloped to convert variable domains of monoclonal antibodies to morehuman form by recombinantly constructing antibody variable domainshaving both rodent and human amino acid sequences. In “humanized”monoclonal antibodies, only the hypervariable CDR is derived from mousemonoclonal antibodies, and the framework regions are derived from humanamino acid sequences.

It is thought that replacement of amino acid sequences in the antibodythat are characteristic of rodents with amino acid sequences found inthe corresponding position of human antibodies will reduce thelikelihood of immune reaction during therapeutic use. A hybridoma orother cell producing an antibody may also be subject to genetic mutationor other changes, which may or may not alter the binding specificity ofantibodies produced by the hybridoma.

It is possible to take monoclonal and other antibodies and userecombinant DNA technology to produce other antibodies or chimericmolecules which retain the antigen specificity of the original antibody,i.e., the molecule has a binding domain. Such techniques may involveintroducing DNA encoding the immunoglobulin variable region or the CDRsof an antibody to the framework regions, constant regions, or constantregions plus framework regions, of a different antibody. See, forinstance, U.S. Pat. No. 5,091,513, and U.S. Pat. No. 6,881,557 which areincorporated herein by this reference.

These antigen binding fragments have binding specificity (bindingdomain) for the antigen, but lack amino acid sequences in the conservedframework region, so they are less likely to elicit an immune responsein a patient. An antigen binding fragment preferably has a bindingdomain provided by one or more antibody variable domains and, mostpreferably, a binding domain of an antibody comprises an antibody lightchain variable region (VL) and an antibody heavy chain variable region(VH).

Examples of antigen binding fragments include, without limitation: (i)the Fab fragment, consisting of VL, VH, CL and CH1 domains; (ii) the“Fd” fragment consisting of the VH and CH1 domains; (iii) the “Fv”fragment consisting of the VL and VH domains of a single antibody; (iv)the “dAb” fragment, which consists of a VH domain; (v) isolated CDRregions; (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments; (vii) single chain Fv molecules (“scFv”), whereina VH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form a binding domain; (viii)bi-specific single chain Fv dimers (see U.S. Pat. No. 5,091,513) and(ix) “diabodies”, multivalent or multispecific fragments constructed bygene fusion (US 2005/0214860). Fv, scFv or diabody molecules may bestabilized by the incorporation of disulphide bridges linking the VH andVL domains. Minibodies comprising a scFv joined to a CH3 domain may alsobe made (S. Hu et al, Cancer Res., 56, 3055-3061, (1996)).

By known means as described above, polyclonal or monoclonal antibodies(humanized, fully human or chimeric), antigen binding fragments andbinding domains may be created that are specific to cardenolides andbufadienolides, their aglycone moieties, or conjugates of any of theforegoing, whether such antigens are isolated from natural sources orare synthetic derivatives or variants of the natural compounds.

Antibodies may be produced from any animal origin, including birds andmammals. Preferably, the antibodies are ovine, murine (e.g., mouse andrat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition,newer technology permits the development of and screening for humanantibodies from human combinatorial antibody libraries. For example,bacteriophage antibody expression technology allows specific antibodiesto be produced in the absence of animal immunization, as described inU.S. Pat. No. 6,946,546 which is incorporated herein by this reference.Theses techniques are further described in: Marks, Bio/Technology10:779-783 (1992); Stemmer, Nature 370:389-391 (1994); Gram et al.,Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992); Barbas et al., Proc.Natl. Acad. Sci., USA, 91:3809-3813 (1994); and Schier et al., J. Mol.Biol. 263:551-567 (1996).

Methods for producing polyclonal antibodies in various animal species aswell as for producing monoclonal antibodies of various types, includinghumanized, chimeric, and fully human, are well known in the art andhighly predictable. In addition, antibodies to various cardiacglycosides, including digoxin, ouabain, bufalin and marinobufagenin, arecommercially available, and methods for producing these antibodies arealso well known and predictable. For example, the following U.S. patentsand patent applications provide enabling descriptions of such methodsand are herein incorporated by reference: U.S. Patent Application Nos.2004/0126828 and 2002/0172677; and U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241;4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720;4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296;5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698;5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091;5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165,464;6,365,157; 6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965;6,849,259; 6,861,572; 6,875,434; and 6,891,024. In addition, allpatents, patent application publications, and other publications citedherein and therein are hereby incorporated by reference in the presentapplication.

It is fully expected that antibodies to a specific cardenolide orbufadienolide will have the ability to neutralize or counteract theeffects of the cardiac glycoside regardless of the animal species,monoclonal cell line or other source of the antibody. For example,although the antibody used in the Biological Examples of the presentapplication was produced in sheep, it is expected that other animalsimmunized with the same or a similar cardenolide or bufadienolide wouldyield polyclonal antibodies effective for the purposes of the presentinvention. Certain animal species may be less preferable for generatingtherapeutic antibodies because they may cause an increased likelihood ofallergic response due to activation of the complement system through the“Fc” portion of the antibody. However, whole antibodies may beenzymatically digested into “Fc” (complement binding) fragment, and intobinding fragments having the binding domain or CDR. Removal of the Fcportion reduces the likelihood that the antigen binding fragment willillicit an undesirable immunological response, and, thus, antibodieswithout Fc may be preferential for prophylactic or therapeutictreatments. As described above, antibodies may also be constructed so asto be chimeric, partially or fully human, so as to reduce or eliminatethe immunological consequences resulting from administering to an animalan antibody that has been produced in, or has sequences from, anotherspecies.

Furthermore, it is known that antibodies to a specific cardenolide orbufadienolide will cross-react with other cardenolides andbufadienolides, although in a less specific manner. Thus, it is believedthat a cross-reactive antibody will have the ability to neutralize orcounteract the effect of the non-specific cardiac glycoside. Forexample, anti-marinobufagenin antibody is known to have cross-reactivity(expressed as a percentage) to various cardiotonic steroids as follows:marinobufagenin (100%); ouabain (0.1%); digoxin (1.0%); digitoxin(3.0%); bufalin (1.0%); proscillaridin (1.0%). Anti-ouabain antibody isknown to have cross-reactivity to various cardiotonic steroids asfollows: ouabain (100%); digitoxin (7.4%); proscillaridin (0.2%);marinobufagenin (0.5%); bufalin (0.03%). Anti-digoxin antibody is knownto have cross-reactivity to various cardiotonic steroids as follows:digoxin (100%); ouabain (0.4%); oubagenin (0.1%); marinobufagenin(0.2%); bufalin (2.7%); cinobufotalin (4.3%); cinobufagin (0.02%). Theseexamples of immunological cross-reactivity are not intended to limit inany manner the scope of the inventions disclosed herein.

If the non-specific binding results in diminished antigen-antibodyaffinity or if the cardenolide or bufadienolide has biopharmaceutical orpharmacokinetic characteristics that differ from the cardiac glycosidethat is specific for the antibody, then a greater amount of antibody maybe required to neutralize or counteract the effects of the non-specificcardenolide or bufadienolide. Nevertheless, it is fully expected and isknown that in vitro antibodies to a specific cardenolide orbufadienolide will have the ability to counteract the sodium pumpinhibition caused by a different cardenolide or bufadienolide. One maydesign appropriate antibody compositions and dosing regimens by takinginto account the level of sodium pump inhibition, the cross-reactivity,binding affinity or avidity, biopharmaceutical and/or pharmacokineticproperties of the specific as compared to the non-specific cardenolideor bufadienolide.

D. Passive Immunity—Neutralizing Antibody Dose Determination.

Determination of an effective antibody composition to neutralizeintoxication by an exogenous antigen usually requires, inter alia, adetermination of the body load of antigen that must be bound orneutralized by the antibody. Factors that influence effective antibodycompositions include, inter alia, known or suspected total body load ofantigen, whether it has reached steady-state equilibrium,bioavailability (free and protein bound), biopharmaceutic andpharmacokinetics of the antigen (e.g. antigen affinity for its receptorand bioactivity), patient weight or volume, history and renal function.Generally, when an antigen reaches equilibrium within the body, antigenconcentration (bound and unbound) in tissues and in extracellular fluidsis reflected by the plasma or serum concentration. Total body load ofthe exogenous antigen generally equals the steady-state serumconcentration of the antigen multiplied by the apparent volume ofdistribution (the fluid volume required to contain the antigen in thebody at the same concentration as in plasma). Thus, the antibodycomposition generally required to neutralize the total body load may bedetermined from the amount of antigen bound by a single unit ofantibody, which is referred to herein as the binding capacity of theunit, multiplied by the total body load of the antigen.

E. Endogenous Factors React with Antibodies to Cardiac Glycosides.

Endogenous factors are known to be immunoreactive to antibodies againsta number of exogenous cardiac glycosides. The body load of an exogenousantigen, which is necessary to determine an effective neutralizingantibody composition, may be determined by immunoassay for the antigen.However, there is no known commercially available antibody orimmunoassay specific for endogenous “digoxin-like” factors. Commerciallyavailable immunoassays have been developed to detect digoxin, ouabain,bufalin and marinobufagenin. These commercially available immunoassayshave been used to detect serum levels of “digoxin,” “ouabain,” “bufalin”or “marinobufagenin” in preeclamptic patients that have never been givenor exposed to any of these exogenous cardenolides or bufadienolides.These levels have previously been believed to accurately represent thelevel of EFs in these patients.

Applicant believes that immunoassays specific for exogenous cardenolidesor bufadienolides, including digoxin immunoassays, have not accuratelydetected levels of EFs present in humans. It is known thatcross-reactivity of EFs to cardiac glycoside antibodies does not alwayscorrelate with the ability of the EFs to cause sodium pump inhibition.Sodium pump inhibition by EFs is often greater than would be expectedbased upon serum levels of EFs (e.g., “digoxin” or “ouabain-likecompound,” “bufalin” or “marinobufagenin”) detected by immunoassay.Also, it is theorized that a substantial portion of EFs, perhaps up to90%, are protein-bound and not detectable by direct measurement withconventional immunoassays. Pullen, M. A., et al., J. Pharm. Exp.Therapeutics 310(10): 319-325 (2004); Valdes, R. and Graves, S. W.,Protein binding of Endogenous Digoxin-immunoactive Factors in HumanSerum and its Variation with Clinical Condition, J. Clin. Endocrinol.Metabol. 60:1135-1143 (1985).

Furthermore, because EFs are likely to be continuously generated in themammal, the total body load of EFs may not necessarily be determined bythe manner used to determine total body load (steady state equilibrium)of an exogenous antigen. In mammals, EFs may not reach “steady state” or“steady state” may be in constant flux. Thus, serum concentrations ofendogenous factors detected by immunoassay for “digoxin”, “ouabain”,“bufalin” or “marinobufagenin” are not likely to accurately measure thetotal body load of EFs at a given time. Thus, it is likely that levelsof endogenous factors detected by known immunoassays represent only aportion of bioactive endogenous factors present in the maternal/fetalcirculation. It is also possible that more than one form or type ofendogenous factor is present in PIH and IUGR and that immunoassays forthese exogenous cardiotonic steroids have not detected certain types ofEFs.

The discrepancy in immunoassay measurements of EFs and the discordancebetween cardiac glycoside immunoactivity and sodium pump inhibitionsuggest that endogenous factors are not digoxin, ouabain, bufalin ormarinobufagenin, but one or more compounds that differ in biological,chemical, physical, biopharmaceutical and/or pharmacokineticcharacteristics from these exogenous cardiac glycosides. Miyagi, H. etal, Ouabain-like Na/K-ATPase Inhibitory Activity of a Plasma Extract inNormal Pregnancy and Pregnancy Induced Hypertension, Japan. J.Pharmacol. 57: 571-581 (1991).

If antibodies to cardenolides and bufadienolides are to be useful indiagnosing or treating the causes or symptoms of PIH and IUGR,conventional antibody compositions to neutralize cardiac glycosideintoxication (e.g., based upon measurements of EFs from immunoassays forexogenous cardiac glycosides, including digoxin, ouabain, bufalin ormarinobufagenin) are not likely to be accurate or effective. Applicanthas surprisingly discovered that digoxin antibody compositions havinghigh doses of digoxin binding capacity are effective to treat thecauses, symptoms and complications of PIH and IUGR.

VII. Therapeutic Antibody Treatment of PIH and IUGR

It is proposed that digoxin antibodies may be given to a patient in atherapeutically effective amount to treat one or more of the causes,symptoms or complications of PIH or IUGR, according to the flow diagramof FIG. 1. Similarly, if prior to onset of symptoms a patient isdetermined to have a propensity for or to be at risk for developing PIHor IUGR, a therapeutically effective amount of digoxin antibody may beadministered for prophylaxis. It should be emphasized, however, that thepresent invention is not limited to any particular composition, dosageor range of dosages of digoxin antibodies, or antibodies from anyparticular source.

A therapeutically effective digoxin antibody composition, whenadministered to a patient exhibiting symptoms or complications of PIH orIUGR, will preferably provide a clinically beneficial effect, and mostpreferably a statistically significant effect, namely the alleviation,amelioration, reduction or inhibition of or improvement in one or moresymptoms or complications of PIH or IUGR, or improvement in thepatient's general condition. A clinically beneficial effect results in abeneficial change from baseline (i.e., before administration of digoxinantibody) for a specific medical parameter. Preferably, a clinicalbeneficial effect results in a reduction in CGI-S or a positive changein CGI-I for the patient. Alternatively, a clinically beneficial effectresults in a systolic blood pressure of 140 or less, a diastolic bloodpressure of 90 or less, or a mean arterial pressure of 106 or less. Astatistically significant effect results in a change in a parameter frombaseline that results in a statistical significance such that “P” isless than or equal to 0.05, whether statistical significance isdetermined with respect to changes in an individual patient or within apopulation of patients. Alternatively, a therapeutically effectiveamount is an amount sufficient to stabilize a symptom of PIH or IUGRsuch that the symptom does not materially worsen.

A therapeutically effective digoxin antibody composition may alsoprovide increased placental and fetal perfusion and/or increased middlecerebral artery (fetal or maternal), umbilical artery or umbilicalvenous flow or reduced constriction. Alternatively, as shown in FIG. 4b, a therapeutically effective composition provides improvement inmiddle cerebral artery (fetal or maternal), umbilical artery orumbilical vein systolic or diastolic blood pressures orsystolic/diastolic ratio (“S/D ratio”), or improvement in the resistanceindex (“RI”) of the middle cerebral artery (fetal or maternal),umbilical artery or umbilical vein.

A therapeutically effective digoxin antibody composition may alsoincrease Na⁺/K⁺ ATPase function and/or Na⁺/K⁺ facilitated nutrienttransport in one or more cell types or tissues, preferably introphoblasts, syncytiotrophoblasts, other placental and fetal cells,vascular endothelial cells, red blood cells, leucocytes, lymphocytes andrenal cells. A therapeutically effective amount may also be the amountnecessary to increase Na⁺/K⁺ ATPase gene expression (e.g. increases inmessenger ribonucleic acid (mRNA) corresponding to a sodium pump gene,which mRNAs are transcribed into polypeptides or proteins that form thesodium pump), or to decrease Na⁺/K⁺ ATPase degradation or recycling inparticular cells or tissues affected by endogenous factors, particularlyvascular endothelial cells, red blood cells, leucocytes, lymphocytes,renal cells, cardiac cells, neural cells, trophoblasts,syncytiotrophoblasts, other placental cells and fetal cells.

Parameters that may be evaluated for or that may indicate therapeuticefficacy of a digoxin antibody composition may include, withoutlimitation: GCI-S or GCI-I; systolic or diastolic blood pressure, ormean arterial pressure; urinary output, urinary protein levels(proteinuria) or creatinine clearance; middle cerebral artery (fetal ormaternal) or umbilical artery or umbilical venous flow, S/D ratio or RI;serum creatinine, LDH, liver enzymes, bilirubin or BUN levels; plateletcount or hemolysis; fetal development (estimated weight for gestationalage and maternal abdominal circumference for gestational age) or fetalbiophysical profile score; peripheral edema, pulmonary edema, cerebraledema and cerebral hemorrhage (including fetal periventricular andintraventricular hemorrhage); scotomata, double or blurred vision,amarousis, cortical blindness, retinal detachment; and neurologicalfunction and/or neurological responsiveness, consciousness, mentalstatus or neurological disturbances (e.g., seizure, hyper reflexia,central nervous system irritability, clonus).

In all methods of the invention, therapeutically effective digoxinantibody compositions may be readily determined by one of ordinary skillin the art by monitoring the patient for signs of absence,stabilization, amelioration, reduction, inhibition, or improvement inone or more indications, symptoms or complications of PIH or IUGR (e.g.,the parameters described in the preceding paragraph), andcorrespondingly maintaining, increasing or decreasing the amount ofdigoxin antibody and/or frequency or manner of administration, asdetermined by clinical judgment.

The composition of the digoxin antibody administered to achieve thedesired therapeutic effect may depend upon a number of factors, forexample, the mode of administration, the recipient's physical condition(including presence of any disease, symptom or syndrome), gestationalage or the anticipated duration of antibody treatment, the existence ofpredisposing factors for any disease, symptom or syndrome (particularlyrisk factors for PIH or IUGR), the severity of the condition or symptomsbeing treated, the patient response to one or more previouslyadministered doses of the antibody and patient response to other drugsor compositions being administered.

The compositions of the invention may be administered to a mammal byany, or a combination of several, means, including, without limitation,oral, intravenous, transmucosal (e.g. nasal, vaginal, etc.), pulmonary,subcutaneous or intradermal or transdermal (injection or infusion orpatch), ocular, buccal, sublingual, intraperitoneal, intrathecal,intramuscular or long term depot preparation. For subcutaneous,intradermal, transdermal, intramuscular or intravenous administration(including intravenous bolus injections or bolus infusions), thecomposition may contain physiologically acceptable solutes, diluents orexcipients including, without limitation, glucose, dextrose, saline,Ringer's or lactated Ringer's solution, Sterile Water for Injection,water or other suitable excipients. For intravenous administration, aloading dose may be administered by intravenous bolus injection or bolusinfusion to achieve a target concentration and may then be followed by asustained intravenous infusion of one or more compositions.

Antibodies to various cardenolides and bufadienolides are well known inthe art, including, without limitation, antibodies to digoxin, ouabainand marinobufagenin. Because digoxin and digitoxin are the primarycardiotonic steroids that are used as therapeutic agents, anti-digoxinantibody products have been developed to treat digoxin or digitoxinoverdose, are well known in the art and have been specifically approvedfor therapeutic use in connection with potentially life-threateningdigoxin and digitoxin intoxication. In rare instances, antibodiesagainst digoxin or digitoxin (or conjugates thereof) have also been used“off label” to treat life-threatening intoxication by other exogenouscardenolides or bufadienolides. Morbidity and Mortality Weekly Report44(46):853-855, 861 (Nov. 24, 1995) (treatment was effective in some,but not all, patients); Eddleston, M. and Warrell, D. A., Management ofAcute Yellow Oleander Poisoning, Q J Med. 92:483-485 (1999) (treatmentwas effective in some, but not all, patients). See, also, Brubacher J R,Ravikumar P R, Bania T, Heller M B, Hoffman R S, Chest 110:1282-1288(1996); Brubacher J R, Lachmanen D, Ravikumar P R, Hoffman R S, Toxicon37:931-942 (1999).

Digoxin immune Fab (ovine) compositions effective for treatinglife-threatening digoxin/digitoxin intoxication are currently marketedby GlaxoSmithKline in the United States under the brand name DIGIBIND®and by Protherics, Inc. under the brand name DIGIFAB™. Digoxin immuneFab products effective for treating life-threatening digoxin/digitoxinintoxication are produced and marketed outside the United States underother brand names. See Clinical studies comparing DIGIBIND® and DIGIFAB™indicate that these products have equivalent pharmacokinetics and theproportion of patients responding to DIGIFAB™ were similar to, andconsistent with, the historical data available for DIGIBIND®.

DIGIBIND® is a sterile lyophilized powder of antigen binding fragments(Fab) derived from specific antidigoxin antibodies raised in sheep.Production of DIGIBIND® involves conjugation of digoxin as a hapten tohuman albumin. Sheep are immunized with this material to produceantibodies specific for the antigenic determinants of the digoxinmolecule. The antibody is then papain-digested, and digoxin-specific Fabfragments of the antibody are isolated and purified by affinitychromatography. These antibody fragments have a molecular weight ofapproximately 46,200 Da. Each vial of DIGIBIND® will bind approximately0.5 mg of digoxin (or digitoxin), and contains 38 mg of digoxin-specificFab fragments plus 75 mg of sorbitol as a stabilizer and 28 mg of sodiumchloride. The vial contains no preservatives. DIGIBIND® is administeredby intravenous injection after reconstitution with Sterile Water forInjection (4 mL per vial), by gentle mixing, to give a clear, colorless,approximately isosmotic solution. Reconstituted product should be usedpromptly. If it is not used immediately, it may be stored underrefrigeration at 2° to 8° C. (36° to 46° F.) for up to 4 hours. Thereconstituted product may be diluted with sterile isotonic saline to aconvenient volume.

DIGIFAB™ is a sterile, purified, lyophilized preparation ofdigoxin-immune ovine Fab (monovalent) immunoglobulin fragments. Thesefragments are obtained from the blood of healthy sheep immunized with adigoxin derivative, digoxin-dicarboxymethoxylamine (DDMA), a digoxinanalogue which contains the functionally essentialcyclopentaperhydrophenanthrene lactone ring moiety coupled to keyholelimpet hemocyanin. The sheep are pathogen free and are from prion-freeherds in Australia. The final product is prepared by isolating theimmunoglobulin fraction of the ovine serum, digesting it with papain andisolating the digoxin-specific Fab fragments by affinity chromatography.These antibody fragments have a molecular weight of approximately 46,000Da. Each vial of DIGIFAB™, will bind approximately 0.5 mg digoxin andcontains 40 mg of digoxin immune Fab, approximately 75 mg of mannitolUSP, and approximately 2 mg sodium acetate (buffering agent). Theproduct contains no preservatives and is intended for intravenousadministration after reconstitution with 4 mL of Sterile Water forInjection USP. The reconstituted product may be added to an appropriatevolume of 0.9% sodium chloride for injection.

DIGIBIND® and DIGIFAB™ are not indicated for milder cases of digitalis(digoxin or digitoxin) toxicity and are only indicated forlife-threatening or potentially life-threatening digoxin or digitoxinintoxication. Although designed specifically to treat life-threateningdigoxin overdose, DIGIBIND® and DIGIFAB™ have also been usedsuccessfully to treat life-threatening digitoxin overdose. However,since human experience is limited and the consequences of repeatedexposures are unknown, DIGIBIND® and DIGIFAB™ are not indicated formilder cases of digitalis toxicity.

DIGIBIND® and DIGIFAB™ are categorized as “Pregnancy Category C” becauseit is not known whether these drugs cause fetal harm when administeredto a pregnant woman or may affect reproductive capacity. According tothe manufacturer's product information, these drugs should be given to apregnant woman only if clearly indicated for life-threatening digoxinintoxication. Also, it is not known whether digoxin antibodies areexcreted in human milk. Because many drugs are excreted in human milk,caution should be exercised when DIGIBIND® or DIGIFAB™ are administeredto a nursing woman.

Clinical indications for administration of DIGIBIND® and DIGIFAB™ do notinclude any condition other than known or suspected life-threateningdigoxin or digitoxin intoxication. For purposes of the invention,life-threatening intoxication (sometimes referred to herein simply as“intoxication” or “intoxicated”) with exogenous cardenolides andbufadienolides means: (a) fatal doses of 10 mg or more in previouslyhealthy adults or 4 mg in previously healthy children; (b) ingestioncausing steady-state serum concentrations greater than 10 ng/mL; or (c)chronic ingestions causing steady-state serum concentrations greaterthan 6 ng/mL in adults or 4 ng/mL in children. DIGIBIND® and DIGIFAB™will interfere with digitalis immunoassay measurements. Thus, standardmeasurement of serum digoxin (or “digoxin”) concentration can beclinically misleading until the antibody is eliminated from the body.

For treatment of intoxication, the composition of DIGIBIND® or DIGIFAB™depends upon the amount of digoxin or digitoxin to be neutralized.However, PIH and MGR are not caused by intoxication from exogenouscardiac glycosides. These conditions are associated with the presence ofendogenous factors produced by the mammal that are distinct from knowncardiac glycosides, including digoxin, ouabain, bufalin andmarinobufagenin. Furthermore, measurements of the concentration ofendogenous factors based upon immunoassays for digoxin, ouabain, bufalinor marinobufagenin are known to be inaccurate and likely underestimatethe amount of endogenous factors present in the mammal.

As demonstrated by Adair (1996) and Goodlin (1988), antibodycompositions effective to treat cardiac glycoside intoxication are noteffective to treat preeclampsia. Therefore, effective antibodycompositions for treating PIH and IUGR cannot be determined based uponantibody compositions that would be effective to neutralize a similarlevel of digoxin in a mammal that has ingested an exogenous cardenolideor bufadienolide. Furthermore, therapeutically effective antibodycompositions for treating PIH and IUGR cannot be determined solely uponindirect measurements of EFs using immunoassays specific for digoxin,ouabain, bufalin or marinobufagenin.

Compositions of digoxin antibody that are effective for treatingdigoxin/digitoxin intoxication may be determined by the followingformula (“Dtox Formula”):Dose (units of antibody)=total antigen body load (mgs)÷digoxin bindingcapacity (mg/unit)

For example, the DIGIBIND® and DIGIFAB™ Dtox Formula used to determinean effective antibody composition for treating intoxication in a patienthaving a known steady-state serum concentration of digoxin/digitoxin is:number of vials=(D×W)÷100, where “D” is the steady-state serum digoxinconcentration and “W” is the patient weight in Kg. Pregnant women withpreeclampsia are known to have serum “digoxin” concentrations ofapproximately ≦1.5, but neither DIGIBIND® nor DIGIFAB™ are approved fortreating any condition other than digoxin intoxication. However, even ifthe EFs in pregnant women were digoxin, neither DIGIBIND® nor DIGIFAB™would be indicated for treating the low levels observed in pregnantwomen. Thus, the tables of adult compositions in the DIGIBIND® andDIGIFAB™ product information do not provide any compositions fortreating serum digoxin/digitoxin concentrations below 1.0 ng/mL, whichwould be comparable to the “digoxin” concentrations normally detected inpregnant women with or without preeclampsia.

Table I identifies putative DIGIBIND® of DIGIFAB™ compositions basedupon the Dtox Formula for treating serum “digoxin” concentrations in therange that endogenous factors have been measured in pregnant women withand without preeclampsia:

TABLE I Dtox Formula for DIGIBIND ® or DIGIFAB ™ Dose (mg digoxinbinding capacity*) Patient Weight Serum “Digoxin” Concentration (ng/mL)(kg) 0.1 0.2 0.4 0.8 1.2 1.6 2.0 40 0.02 0.04 0.08 0.16 0.24 0.32 0.4 600.03 0.06 0.12 0.24 0.36 0.48 0.6 70 0.035 0.07 0.14 0.28 0.42 0.56 0.780 0.04 0.08 0.16 0.32 0.48 0.64 0.8 100 0.05 0.10 0.20 0.40 0.60 0.801.0 120 0.06 0.12 0.24 0.48 0.72 0.96 1.2 140 0.07 0.14 0.28 0.56 0.841.12 1.4 *[weight × digoxin concentration/100] × 0.5 mg digoxin bindingcapacity per vial

Experiments of Goodlin (1988) and Adair (1996) have used the DtoxFormula to determine DIGIBIND® compositions to administer topreeclamptic patients. Goodlin administered a composition of 10 mgDIGIBIND® for a total of 0.13 mgs digoxin binding capacity, resulting ina dose of 0.001 mg digoxin binding capacity per Kg patient body weight(115 Kg patient). Adair administered a composition of 29 mg antibodyhaving a total of 0.38 mg digoxin binding capacity, but the patientweight was not published. Applicant-author's notes indicate the patientweight was 65 Kg. Therefore, Adair administered digoxin binding capacityof 0.0058 mg per Kg patient weight.

Goodlin achieved only a transient, precipitous decrease in mean bloodpressure immediately after administration of the antibody, and theresults may have been due, in part, to concurrent administration of anantihypertensive drug along with albumin. Adair achieved a gradualreduction in mean arterial pressure, most notably 12 hours afteradministration of the antibody, but with a concomitant and significantworsening of proteinuria. In both cases the pregnancy was terminatedprior to term and the fetuses did not survive. Thus, the Dtox Formulaand the antibody compositions administered by Goodlin and Adair haveproven to be ineffective in treating preeclampsia. Furthermore, sinceAdair used a dose of digoxin antibody greater than that used by Goodlinand that higher dose resulted in a significant worsening of proteinuria,it would be expected that even higher doses would have similar orgreater adverse side effects. Thus, one would not expect higher doses ofdigoxin antibody to be therapeutically effective to treat PIH or IUGR.

Surprisingly, Applicant has discovered that symptoms and complicationsof PIH and IUGR may be effectively treated by administration of adigoxin antibody compositions having as an active ingredient a high doseof digoxin binding capacity, and that such high dose compositions do notcause a worsening of proteinuria. Therapeutically effective high dosedigoxin antibody compositions preferably comprise an amount of digoxinantibody sufficient to provide more than 0.006 mgs digoxin bindingcapacity per Kg patient weight when symptoms or complications of PIH orIUGR are present. Preferably, a high dose composition has digoxinbinding capacity that is between ten-fold and one hundred-fold thedigoxin binding capacity that would be provided in a compositionaccording to the Dtox Formula. Most preferably, a high dose compositioncomprises approximately ten-fold the digoxin binding capacity that wouldbe provided in a composition according to the Dtox Formula for treatingintoxication evidenced by a similar serum digoxin concentration.Therapeutic compositions less than ten-fold the composition providedaccording to the Dtox Formula are also effective, provided suchcompositions have more than 0.006 mg digoxin binding capacity per Kgpatient weight if the patient manifests clinical symptoms of PIH orIUGR. Therapeutically effective compositions for prophylaxis may alsohave less than 0.006 mg digoxin binding capacity per Kg patient weight.

It should be emphasized that the present invention is not limited todigoxin-immune Fab (ovine), DIGIBIND® or DIGIFAB™, or to any particularbrand or formulation of digoxin antibody, but encompasses allantibodies, including antigen binding fragments and binding domains,that react immunologically with or bind digoxin. Further the inventionis not limited to any particular high dose digoxin antibody composition,dosing method or regimen.

The compositions of the invention comprise a single application dose. Asingle application dose includes the composition when given (a) entirelyin a single administration or (b) when given in aliquots as multipleadministrations over a period of time, or (c) when administeredcontinuously over a period of time (e.g., continuous infusion, slow ortime release transdermal administration, slow-release or time-releasetablets or capsules or caplets). When a composition is given in aliquotsover a period of time or is continuously administered over a period oftime, preferably the period of time is thirty-six hours or less, morepreferably twenty-four hours or less and most preferably twelve hours orless.

The high dose digoxin antibody composition preferably has betweenapproximately 0.5 and 100 mg digoxin binding capacity, more preferablybetween approximately 1.0 and 50 mg digoxin binding capacity, and mostpreferably between approximately 1.0 and 15 mg digoxin binding capacity.Sample pharmaceutical compositions of high dose digoxin antibodiesappropriate for typical human weights are described in Table II.A andthe preferred compositions are provided in Table II.B (approximately 10×DIGIBIND®/DIGIFAB™ Dtox compositions), compositions). Table IIIrepresents the preferred compositions of the current formulations ofDIGIBIND® or DIGIFAB™ (approximately 10× DIGIBIND®/DIGIFAB™ Dtoxcompositions), expressed as number of vials. For human or other mammalweights that are greater than 140 Kg, an appropriate composition may bedetermined by dividing the mammal's weight by 100 and multiplying theresult by a digoxin binding capacity within the range of digoxin bindingcapacities provided in Table II.A. or II.B. for a mammal weighing 100Kg. For example, if a mammal weighs 500 Kg and has a serum “digoxin”concentration of 1.0 ng/mL, then an appropriate digoxin antibodycomposition according to Table II.B. is: 500/100×5.0 mg digoxin bindingcapacity=25 mg digoxin binding capacity (or 50 vials of either DIGIBIND®or DIGIFAB™ in the formulations described in the notes to Table III).

TABLE II.A Sample Digoxin Antibody Compositions for PIH and IUGR (mgdigoxin binding capacity) Patient Weight Serum “Digoxin” Concentration(ng/mL) (kg) ≦0.1* 0.2 0.4 0.8 1.2 1.6 2.0 40 0.25 0.40 0.50 0.60 0.700.80 1.0 60 0.40 0.45 0.50 0.55 0.75 1.0 1.2 70 0.45 0.50 0.55 0.65 0.851.2 1.4 80 0.55 0.55 0.60 0.75 1.0 1.3 1.6 100 0.65 0.75 0.80 0.80 1.21.6 2.0 120 0.80 0.85 .90. 1.0 1.5 2.0 2.4 140 0.90 1.2 1.3 1.5 1.8 2.42.8 *compositions are approximately 0.0065 mg digoxin binding capacityper Kg patient weight.

TABLE II.B Preferred Digoxin Antibody Compositions** for PIH and IUGR(mg digoxin binding capacity) Patient Weight Serum “Digoxin”Concentration (ng/mL) (kg) ≦0.1* 0.2 0.4 0.8 1.2 1.6 2.0 40 0.5 0.5 1.01.5 2.5 3.5 4.0 60 0.5 0.5 1.5 2.5 3.5 5.0 6.0 70 0.5 1.0 1.5 3.0 4.55.5 7.0 80 0.75 1.0 1.5 3.5 5.0 6.5 8.0 100 0.75 1.0 2.0 4.0 6.0 8.010.0 120 1.0 1.0 2.5 5.0 7.0 9.5 12.0 140 1.0 1.5 3.0 5.5 8.5 11.0 14.0*compositions suggested for initial prophylactic administration **mostcompositions are approximately 10x the dose of DIGIBIND ®/DIGIFAB ™ asdetermined by the Dtox Formula

TABLE III Preferred Digoxin Antibody Compositions ofDIGIBIND ®/DIGIFAB ™ for PIH and IUGR (in # of vials) Patient WeightSerum “Digoxin” Concentration (ng/mL) (kg) ≦0.1* 0.2 0.4 0.8 1.2 1.6 2.040 1 V 1 V 2 V 3 V  5 V  7 V  8 V 60 1 V 1 V 3 V 5 V  7 V 10 V 12 V 70 1V 2 V 3 V 6 V  9 V 11 V 14 V 80 1.5 V   2 V 3 V 7 V 10 V 13 V 16 V 1001.5 V   2 V 4 V 8 V 12 V 16 V 20 V 120 2 V 3 V 5 V 10 V  15 V 20 V 24 V140 2 V 3 V 6 V 12 V  17 V 23 V 28 V *compositions appropriate forinitial prophylactic administration V = vials, in most casesconstituting approx. 10x Dtox Formula DIGIBIND: 38 mg antibody/vial, 0.5mg digoxin binding capacity/vial DIGIFAB: 40 mg antibody/vial, 0.5 mgdigoxin binding capacity/vial

In yet another embodiment, the therapeutic composition comprises anamount of digoxin antibody sufficient to provide between greater than0.006 mg and approximately 10.0 mg digoxin binding capacity per Kgpatient body weight, more preferably between approximately greater than0.006 and approximately 5.0 mgs digoxin binding capacity per Kg patientbody weight, even more preferably between approximately 0.01 and 1.0 mgdigoxin binding capacity per Kg patient body weight, and most preferablybetween approximately 0.01 and 0.5 mg digoxin binding capacity per Kgpatient body weight.

The compositions of Tables II.A, II.B and III are preferred compositionsonly, and compositions appropriate for any particular patient weight orserum “digoxin” concentration may have digoxin binding capacity greaterthan or less than the above amounts, provided that compositions to treatpatients manifesting clinical symptoms of PIH or IUGR comprise high dosecompositions having greater than 0.006 mg digoxin binding capacity perKg patient weight (e.g. Table II.A, compositions for serum “digoxin”levels of ≦0.1 ng/mL). For serum “digoxin” concentrations fallingbetween the specific concentrations identified in Tables II.A., II.B andIII, in most cases the composition should be given in an amount that isintermediate between the amounts for the next lower and next higherserum “digoxin” concentration for the patient's weight. Similarly, forpatient weights falling between those listed in Tables II.A, II.B andIII, in most cases the dose should be given for the next highest weightfor the particular serum “digoxin” concentration. However, if anintermediate amount may be obtained, the intermediate amount ispreferable. For example, if serum “digoxin” concentration is 0.8 ng/mL(whether measured or assumed to be present based upon severity of one ormore symptoms) for a patient weight of 115 Kg, a therapeutic compositioncomprising 9 vials of DIGIBIND® or DIGIFAB™ (Table III) would beadministered as a single application dose.

For digoxin antibody formulations (i.e., dosing units), including,without limitation, DIGIBIND® and DIGIFAB™, that have greater or lesserdigoxin binding capacity than the DIGIBIND and DIGIFAB formulations ofTable III (0.5 mg per vial), antibody compositions should be adjusted toprovide digoxin binding capacity that is equivalent to the digoxinbinding capacity of the compositions (# vials) described in Table III.

Serum “digoxin” concentration should be obtained before initialadministration of digoxin antibody if at all possible. After initialadministration, measurements of serum “digoxin” concentration may bemisleading because digoxin antibodies are known to interfere withstandard digoxin immunoassay and so digoxin antibodies are also expectedto interfere with measurement of EFs by digoxin immunoassay. Although itis preferable to measure serum “digoxin” concentration before initialadministration of digoxin antibody, it is not necessary to quantify apatient's serum “digoxin” concentration in order to determine atherapeutically effective composition. If serum “digoxin” concentrationcannot be readily determined, the subject may be given an initialcomposition commensurate with the severity of the symptoms of PIH orIUGR. Generally, mild symptoms may initially be treated with atherapeutic composition at the lower end of the therapeutic range forthe patient's weight, according to Table II.A, Table II.B., or TableIII. Preferably, compositions for mild symptoms and prophylactictreatment are initially selected from the lower range of compositions ofTable II.A. The patient should be routinely monitored throughout thecourse of treatment for improvement, amelioration, stabilization orworsening of symptoms or complications of PIH and IUGR. If the patientresponse to a composition is adequate in the clinical judgment of thetreating physician, then the composition may be repeated as often asneeded to maintain the desired response. If an adequate response is notachieved with a composition or a greater response is desired, then acomposition having a greater amount of the active ingredient (digoxinbinding capacity) should be given in one or more subsequentadministrations.

More severe symptoms should be initially treated in the mid-range of thecompositions for the patient's weight according to Table II.A, TableII.B or Table III. Again, the patient should be routinely monitored foramelioration, improvement, stabilization or worsening of symptoms orcomplications of PIH and IUGR, so that the composition may be adjusted(active ingredient decreased or increased) as determined by clinicaljudgment. For illustration purposes only, and without limitation, a 70Kg patient presenting with symptoms of severe preeclampsia may beinitially administered 3 vials of DIGIBIND® or DIGIFAB™ or digoxinantibody of another formulation having approximately 1.5 mg digoxinbinding capacity, but if eclampsia is present the patient may beinitially administered a composition of between 4 and 6 vials ofDIGIBIND® or DIGIFAB™ or a digoxin antibody composition of anotherformulation or antibody species having between approximately 2.0 and 3.0mg digoxin binding capacity.

It is known that pregnant women (normotensive and with PIH) have serumconcentrations of endogenous factors in the range of ≦0.1 ng/mL to 1.5ng/mL (as measured by digoxin immunoassay). It is also known that levelsof endogenous factors (as measured by digoxin immunoassay) increasethroughout pregnancy (i.e., third trimester>second trimester>firsttrimester). See, Beyers A D, et al., The possible role of endogenousdigitalis—like substance in the causation of pre-eclampsia, SA MedicalJournal 65:883-885 (1984); Craig H R, et al., Binding of EndogenousDigoxin-like Immunoreactive Factor to Serum Proteins During Normal andHypertensive Pregnancy, J. Clin. Immunoassay 14(4):245-250 (1991);Lopatin D A, et al., J Hypertension 17(8): 1179-1187 (1999). Thus, forpurposes of the invention a serum “digoxin” concentration may also beselected based upon serum “digoxin” concentrations generally known to bepresent in pregnant patients, and preferably serum “digoxin”concentrations within the range generally known to be present inpatients with PIH. Most preferably, the serum “digoxin” concentrationwill be based upon the range generally known to be present in PIH, willbe selected based upon the state of pregnancy (first, second or thirdtrimester), risk factors for PIH or IUGR (in kind or number), or symptomseverity and/or complications commensurate with those of the patientbeing treated. For example, it is known that patients experiencingsymptoms of severe preeclampsia have serum “digoxin” concentrationsgreater than or equal to 0.3-0.4 ng/mL during the second trimester ofpregnancy. Thus, a patient in her second trimester presenting withsymptoms of severe preeclampsia may initially be given a compositioncommensurate with 0.3-0.4 ng/mL, as provided in Tables II.A, II.B orIII.

Preferably, the patient is initially given a therapeutic compositionhaving the lowest amount of active ingredient (digoxin binding capacity)appropriate for the patient's weight, as determined by clinicaljudgment. Thereafter, one or more subsequent compositions having greateramounts of the active ingredient may be administered to achieve thedesired improvement in symptoms or complications of PIH or IUGR.However, in situations where the patient's symptoms or complications arepotentially life-threatening, it may be preferable to initiallyadminister a therapeutic composition having an amount of the activeingredient greater than the lowest therapeutic composition for thepatient's weight.

The digoxin antibody of the composition is any antibody, bindingfragment or binding domain that reacts immunologically with or bindsdigoxin, and preferably an antibody, binding fragment or binding domainspecific for a cardenolide or bufadienolide, including, withoutlimitation, anti-ouabain antibody, anti-marinobufagenin antibody,anti-bufalin antibody and anti-digoxin antibody. Methods for quantifyingrelative cross-reactivity of an antibody for a different antigen orepitope are well known to those skilled in the art. See, for example,Fedorova et al., Journal of Hypertension 23(4):835-842 (2005) andreferences therein. Generally, if an antibody is known to cross-reactwith digoxin, the cross-reactivity provides a basis to formulate acomposition of such antibody to provide the digoxin binding capacity ina therapeutically effective amount as described above. For example, if aformulation of marinobufagenin antibody has 0.5 mg marinobufageninbinding capacity and a cross-reactivity to digoxin of 10%, then the samecomposition will have approximately 0.05 mg digoxin binding capacity.

The composition may be comprised of whole digoxin antibody, bindingfragments, binding domains, or a combination thereof. The digoxinantibody may be polyclonal, monoclonal, chimeric, humanized or fullyhumanized and may be generated from any known antibody source,including, without limitation, sheep, goat, horse, chicken, rabbit,mouse, mammalian cells lines, bacteria or yeast. The digoxin antibodysource is preferably ovine (produced in sheep, or generated orconstructed, in whole or in part, from polypeptides, proteins or nucleicacids of ovine origin). Preferably, the digoxin antibody is digoxinimmune Fab.

In another embodiment, the composition comprises a therapeuticallyeffective amount that is sufficient to cause a clinically beneficial ora statistically significant reduction in systolic blood pressure,diastolic blood pressure or mean arterial pressure. In alternateembodiments, the composition has a therapeutically effective amountsufficient to cause a clinically beneficial or a statisticallysignificant decrease in proteinuria or a decrease in a patient's serumcreatinine level or in a liver enzyme level. Alternatively, thecomposition has a therapeutically effective amount sufficient to cause aclinically beneficial or a statistically significant increase in urinaryoutput or creatinine clearance.

In another embodiment, the therapeutic composition comprises an amountof digoxin antibody sufficient to cause a reduction in vasospasm,vasoconstriction, exaggerated myocardial function or intravascularvolume contraction.

In yet another embodiment, the therapeutic composition comprises anamount of digoxin antibody sufficient to cause an improvement in arteryor venous flow, preferably in a middle cerebral artery (fetal ormaternal), an umbilical artery or an umbilical vein. Preferably, theamount is sufficient to cause a reduction in or substantialnormalization of the S/D ratio or the RI of an artery or vein, or toimprove diastolic flow of an artery or vein (e.g., improve the diastoliccomponent of the S/D ratio). Most preferably, the reduction,normalization or improvement is in a middle cerebral artery (fetal ormaternal), an umbilical artery or an umbilical vein.

In another embodiment, the composition is therapeutically effective tocause a clinically beneficial or a statistically significant decrease inmean blood pressure (systolic, diastolic or MAP) taken over a period oftime. In an alternate embodiment, the therapeutic composition iseffective to cause a clinically beneficial or a statisticallysignificant reduction in systolic blood pressure, diastolic bloodpressure or mean arterial pressure that commences at a time that is lessthan four hours after beginning administration of the composition. In analternate embodiment, the reduction in systolic or diastolic bloodpressure or mean arterial pressure commences at a time that is less thanfour hours after beginning administration of the composition andcontinues for a period of time that is more than four hours afterbeginning administration of the composition.

While the above therapeutic compositions have been provided, deviationsor modifications may be used, provided that the composition has morethan 0.006 mg digoxin binding capacity per Kg patient body weight if thepatient manifests clinical symptoms of PIH or IUGR. Furthermore, thecompositions simply define a lower limit for treatment of gestationalhypertension, preeclampsia, eclampsia and IUGR. Therefore, compositionshaving higher doses of digoxin binding capacity than the compositionsprovided in Tables II.A., II.B. and III are also expected to beeffective and are also encompassed within the present invention.

A method is also provided for treating gestational hypertension,preeclampsia, eclampsia or IUGR, by administering a therapeuticallyeffective amount of a high dose digoxin antibody composition to apregnant mammal.

In one embodiment, the method includes the step of administering betweengreater than 0.006 mg and approximately 10.0 mg of digoxin bindingcapacity per kilogram of patient body weight. Preferably, the methodcomprises administration of a digoxin antibody composition havingdigoxin binding capacity between greater than 0.006 mg and approximately5.0 mg per kilogram of patient body weight, more preferably betweenapproximately 0.01 mg/Kg and 1.0 mg/Kg patient body weight, and mostpreferably between approximately 0.01 mg and 0.5 mg/Kg patient bodyweight.

In yet another embodiment of the invention (FIG. 1), the method includesevaluating a patient for one or more risk factors, diagnosticindicators, causes, symptoms or complications of gestationalhypertension, preeclampsia, eclampsia or IUGR. The method may alsoinclude determining that the patient does evidence a risk factor,diagnostic indicator, cause, symptom or complication of PIH or IUGR, andthen administering a therapeutically effective composition of high dosedigoxin antibody commensurate with the clinical assessment.

Administration of the digoxin antibody composition for prophylaxis(e.g., before onset of diagnostic indications or the manifestation ofclinical symptoms or complications of PIH or IUGR) is preferablycommenced between approximately 14 and 20 weeks' gestation and may becontinued throughout the pregnancy for so long as may be medicallydesirable, advisable or necessary until such time as the patient'ssymptoms worsen and no longer respond to a therapeutic high dose digoxinantibody composition, or, in the treating physician's clinical judgment,delivery of the fetus is desirable, advisable or necessary. Compositionsfor prophylaxis may have digoxin binding capacity less than 0.006 mg/Kgpatient weight.

In an alternate embodiment, prophylactic administration of a high dosedigoxin antibody composition may begin before or after 14 to 20 weeks'gestation, at any time prior to onset of diagnostic indications or themanifestations of clinical symptoms or complications of PIH or IUGR, orat any time after diagnostic tests or patient evaluation indicates thepatient is at risk for developing or the likely emergence of gestationalhypertension, preeclampsia, eclampsia or intrauterine growthrestriction.

Preferably, a therapeutically effective amount of a high dose digoxinantibody composition is given for prophylaxis when the patient has oneor more risk factors for PIH or IUGR, including those risk factors fromamong the group risk factors consisting of first pregnancies, pregnantwomen under the age of 20 or over the age of 35, women of black race,women who conceive through IVF, women who have a prior pregnancy with adifferent partner or have a long interval between pregnancies,multi-gestational pregnancies, women with a personal or family historyof PIH or IUGR, women who are of higher than normal weight or body massindex (>25) prior to pregnancy, women who have thrombophilia riskfactors, and women with a personal or familial history of polycysticovarian syndrome, diabetes, hypertension, renal (kidney) disease,rheumatoid arthritis, systemic lupus erythematosus, other autoimmunedisease or multiple sclerosis.

In yet another embodiment of the method, administration of the high dosedigoxin antibody composition is given intravenously, as a continuousinfusion or in the form of a bolus infusion or a bolus injection. Acontinuous infusion is preferably given over a period of 36 hours orless, more preferably over 24 hours or less, and most preferably overtwelve hours or less. A bolus infusion is preferably given byadministration of the dose by infusion over a period longer than 10minutes and less than or equal to 1 hour, preferably over thirtyminutes. A bolus injection is preferably given by administration of thedosage over a period of ten minutes or less. Alternatively, the digoxinantibody may be administered via intramuscular or subcutaneous ortransdermal injection or infusion.

In yet another embodiment of the method, administration of a high dosedigoxin antibody composition is repeated. The same high dose digoxinantibody composition may be repeated. Alternatively, the repeatedadministration is of a high dose digoxin antibody composition that isgreater than or less than a previously administered composition. Thecomposition to be given in any repeat administration is determined byclinical judgment based upon patient symptoms, responsiveness to one ormore prior high dose digoxin antibody compositions (e.g., the medicalparameters described above that determine therapeutic effectiveness) orbased upon other appropriate medical criteria.

In an alternate embodiment, a repeat administration of a high dosedigoxin antibody composition is given between approximately one andthirty-six hours after a prior administration of a high dose digoxinantibody, preferably between approximately one and twenty-four hours,more preferably between one and fifteen hours, even more preferablybetween three to eight hours, and most preferably at approximately sixhours.

In a preferred embodiment, the high dose digoxin antibody composition isrepeatedly administered on a fixed schedule, the fixed schedule beingbetween approximately every one and every thirty-six hours, morepreferably between approximately every one and every twenty-four hours,even more preferably between every one and fifteen hours, yet even morepreferably between every three and every eight hours, and mostpreferably approximately every six hours.

Therapeutically effective high dose digoxin antibody compositions may besufficient to reduce blood pressure to such an extent thatantihypertensive agents are not required. However, a high dose digoxinantibody composition may also be administered with antihypertensiveagents. In one embodiment, the method comprises administration of a highdose digoxin antibody and a therapeutically effective amount of anantihypertensive agent. The antihypertensive agent may be administeredprior to, subsequent to, or concurrent with the administration of thehigh dose digoxin antibody composition. Preferably the antihypertensiveagent is selected from among the group consisting of hydralazine,nifedipine, sodium nitroprusside, 1-methyldopa (e.g., Aldomet®),atenolol and labetalol. Dosing amounts and regimens for administeringthese antihypertensive agents during pregnancy are well known in theart. Generally, a therapeutically effective amount of hydralazine isbetween 10 mg and 100 mg, preferably hydralazine is administered orallyas 10 mg, 25 mg, 50 mg or 100 mg tablets. Alternatively, hydralazine maybe administered intravenously. Preferably, hydralazine is administeredat least once per 24 hours. Generally, a therapeutically effectiveamount of nifedipine is between 10 mg and 100 mg. Preferably, a dose ofnifedipine is administered orally at least once per 24 hours. Generally,a therapeutically effective amount of methyldopa (e.g., Aldomet) isbetween 250 and 3000 mg. Methyldopa may be administered orally,preferably as 15 mg, 25 mg, 30 mg, 50 mg, 125 mg, 250 mg or 500 mgtablets. Alternatively, methyldopa may be administered intravenously.Generally, a therapeutically effective amount of atenolol is between 5mg and 100 mg, preferably between 25 and 100 mg. Preferably, a dose ofatenolol is administered orally at least once per 24 hours. Generally,an effective amount of sodium nitroprusside is administeredintravenously between 0.1 μg/Kg/minute and 0.6 μg/Kg/minute.

Preferably, the antihypertensive is labetalol. The patient is preferablyadministered 20 mg labetalol by intravenous injection over two minutes.In an alternate embodiment, the patient is repeatedly administeredlabetalol by injection, preferably in doses between approximately 20 mgand 80 mg, and most preferably approximately every ten to fifteenminutes. Alternatively, labetalol may be administered as a continuousinfusion. Preferably 20 to 80 mg labetalol is infused at a rate of 1.0mg/minute. The labetalol dose may be adjusted as indicated by patientresponse and clinical judgment. Preferably, labetalol is given in amaximum aggregate dose of 240 mg.

In yet another embodiment, the method further comprises administrationof a therapeutically effective amount of a pharmaceutical agent toimprove fetal development or to limit the adverse effects of prematuredelivery on the neonate. Preferably the pharmaceutical agent is acorticosteroid to enhance fetal lung maturity, to reduce the incidenceor severity of respiratory disease in the neonate or to lessenperiventricular or intraventricular hemorrhage or neurological damage inthe neonate. Most preferably the corticosteroid is bethamethasone ordexamethasone.

In yet another embodiment, the corticosteroid is administered at leasttwice during a consecutive period of forty-eight hours. Corticosteroidsare even more preferably given by intramuscular injection of 12 mgsbethamethasone every twenty-four hours or intramuscular injection of 6mgs dexamethasone every twelve hours, during a period of at leastforty-eight consecutive hours.

Corticosteroids are preferably administered either at least 30 minutesprior to or at least 30 minutes after the administration of a digoxinantibody composition. In yet another embodiment, the corticosteroid isadministered at least 12 hours prior to administration of a first doseof digoxin antibody. In another embodiment, the second dose ofcorticosteroid is administered at least 6 hours prior to a subsequentdose of digoxin antibody.

In another embodiment the method further comprises administration of aneffective amount of an anti-convulsant, preferably magnesium sulfate orphenytoin. Generally, a therapeutically effective amount of phenytoin isbetween 10 and 100 mg. Preferably, phenytoin is administered at leastonce per 24 hours, and may be administered two or three times per 24hours.

In yet another embodiment, an effective amount of magnesium sulfate maybe administered prior to, subsequent to, or concurrent withadministration of the digoxin antibody composition. Preferably,magnesium sulfate (100 mL of a 0.5 gm/mL solution, or 50 gm) is added to400 mL normal saline for a total volume of 500 mL. Administration ispreferably by intravenous infusion over 30 minutes. Administration ofmagnesium sulfate may be repeated periodically or administeredcontinuously. The patient is preferably administered a loading dose ofmagnesium sulfate intravenously, most preferably the loading dose is 6gm or 60 mL of a 0.1 gm/mL solution. Preferably, after administration ofa loading dose of magnesium sulfate the dosing is maintained at 2 gm/hr(20 mL/hr).

In yet another embodiment, the antibody composition is administered incombination with intravenous fluids, preferably lactated Ringer'ssolution. Most preferably, intravenous fluids are combined withmagnesium sulfate for administration at a total fluid rate between 40mL/hr and 250 mL/hr, preferably 125 mL/hour. Intravenous fluids may beadjusted during the course of digoxin antibody treatment as determinedby clinical judgment.

In an alternate embodiment of the method, one or more repeat doses of ahigh dose digoxin antibody composition are independently selected (i.e.,a prior composition is not automatically repeated) prior toadministration, allowing the treatment to be adjusted over time basedupon the individual patient condition, response to prior digoxinantibody compositions (e.g., the medical parameters that determineeffectiveness, as described above) and other relevant medical or dosingcriteria.

In yet another embodiment of the method, during a consecutive period ofthirty hours of less, the subject is administered one or more high dosedigoxin antibody compositions having an aggregate digoxin bindingcapacity of at least 0.032 mg per kilogram of patient weight.Alternatively, during a period of thirty hours or less the patient isadministered one or more high dose digoxin antibody composition havingan aggregate digoxin binding capacity between approximately 0.032 mg andless than or approximately equal to 0.171 mg per kilogram of thepatient's weight. In another embodiment, the aggregate digoxin bindingcapacity administered in one or more high dose digoxin antibodycompositions over a consecutive 30 hour period or less is betweenapproximately 0.066 mg and approximately 0.171 mg per kilogram of thepatient's weight. In yet another embodiment, the aggregate digoxinbinding capacity administered in one or more compositions over a 30 hourperiod or less is between approximately 0.081 mg and approximately 0.171mg per kilogram of the patient's body weight. In yet another embodiment,the aggregate digoxin binding capacity administered in one or morecompositions over a 30 hour period or less is between approximately0.122 mg and approximately 0.171 mg per kilogram of the patient'sweight.

Another embodiment of the invention provides a method for treatinggestational hypertension, preeclampsia, eclampsia or intrauterine growthrestriction, comprising the step of administering at least onecomposition of a digoxin antibody from among the range of compositionsdescribed in Table II.A., Table II.B or Table III, so as to provide acomposition having greater than 0.006 digoxin binding capacity perkilogram of the patient's weight. In yet another embodiment, thecomposition is selected from any one of Tables II.A, II.B or Table IIIbased upon the patient's weight.

In yet another embodiment of the method, the high dose digoxin antibodycomposition is given to a patient to treat intrauterine growthrestriction. The umbilical artery flow velocity in a preeclampticpatient is illustrated in FIG. 3. Preeclamptic vasoconstriction reducesthe blood flow velocity through arteries and veins, particularly themiddle cerebral artery (maternal or fetal), an umbilical artery or theumbilical vein. Reduced flow, particularly in the umbilical arteryresults in the flow velocity “bottoming out” during diastole (indicatedin FIG. 3 as “C” and in FIG. 4 a) such that little or no blood isflowing to the fetus and/or placenta. This results in a highsystolic/end diastolic (“S/D”) ratio and a high resistance index (“RI”).Reduction in artery flow or venous flow, particularly in the middlecerebral artery or an umbilical artery or the umbilical vein, restrictsoxygenation, fluid exchange and nutrition, potentially causing growthrestriction of the fetus. Administration of a high dose digoxin antibodycomposition eases vasoconstriction in arteries and veins, and as shownin FIG. 4 b, and results in a decrease in or substantial normalizationof the S/D ratio and/or RI of a middle cerebral artery (maternal orfetal), an umbilical artery or the umbilical vein.

In one embodiment, the method includes administration of a high dosedigoxin antibody composition to substantially normalize the S/D ratio ofthe middle cerebral artery (maternal or fetal), or to improve the S/Dratio of an umbilical artery or the umbilical vein. In yet anotherembodiment, the high dose digoxin antibody composition is administeredto substantially normalize the RI of the middle cerebral artery(maternal or fetal), or to improve the RI of an umbilical artery orumbilical vein.

While the above compositions have been provided, deviations ormodifications may be used. Again, the therapeutically effective highdose compositions described above simply define a lower limit ofcompositions providing greater than 0.006 mg digoxin binding capacityper kilogram patient weight when a patient manifests clinical symptomsof PIH or IUGR; therefore, effective compositions having digoxin bindingcapacity higher than this lower limit, or greater than any of theexamples specifically provided herein, are also expected to be effectiveand so all such compositions are also encompassed within the presentinvention. Similarly, high dose compositions for prophylaxis maycomprise digoxin binding capacity that is less than 0.006 mg per Kgpatient weight, and although not specifically provided in the Tables andexamples provided herein, all such compositions are encompassed withinthe present invention.

VIII. Biological and Prophetic Examples

The method is further illustrated in the following non-limitingexamples.

Biological Example #1

A 16-year-old 70 Kg primigravida at 29 weeks 5/7 days presented with apresumed diagnosis of eclampsia. Her prenatal course had beenuncomplicated until the day of admission. Her past medical history wasnegative for chronic illness. The patient had complaints of scotomata,persistent headache, and reported seizure activity for two episodesprior to hospital arrival and one on the labor deck. Initial evaluationrevealed an alert patient with obvious postictal behavior. Her bloodpressure was elevated to approximately 160/110s and otherwise had stablevital signs. Physical examination revealed arterio-venous nicking onopthoscalmic exam, 4+ edema of the lower extremities and obvious facialand upper extremity edema. Deep tendon reflexes were 3+ with 2 beats ofclonus. The remainder of the complete exam was within normal limits. Labevaluation of the urine noted proteinuria on qualitative analysis of 2+and a urine specific gravity of 1.020. Serum chemistry revealedhyperuricacidemia at 8.1, elevated serum creatinine of 1.0, BUN of 6,and otherwise normal electrolytes and liver enzymes. The complete bloodcount revealed a platelet count of 429,000, white count of 10.4 and ahemoglobin and hematocrit of 12.0 and 35.6, respectively. The ammonialevel was 3 and coagulation studies were normal. Urine drug screeningwas negative for substances of abuse including cocaine andmethamphetamines. CT scanning of the maternal head failed to reveal anypathological abnormalities.

Ultrasound examination revealed a single gestation with a breechpresentation. The estimated gestational age was consistent with thepreviously determined age of 29 weeks with an estimated fetal weight of1331 grams. The amniotic fluid index was 5.42 cm and fetal breathing,movement, and tone were noted to be present. Doppler flow studies of theumbilical artery revealed an elevated S/D ratio of 5.6, RI of 0.82, andminimal diastolic flow. No anatomical abnormalities were noted on exam.Fetal cardiotocograph revealed a baseline of 135 with minimalbeat-to-beat variability. Occasional non-repetitive decelerations werenoted with good recovery. They were deemed to be non-ominous.

The patient was placed on intravenous magnesium sulfate. Central venousaccess and arterial line placement were performed. After informeddiscussion with the immediate family including the option to stabilizewith compassionate off label use of fragmented antibody to digoxin andto administer corticosteroids for fetal benefit, the patient's motherprovided consent.

Standard preeclampsia monitoring every hour was followed with one on onenursing in the intensive care unit. Based upon a level of 0.4 ng/mLserum “digoxin” concentration (believed to be present but notquantified), digoxin immune Fab (0.5 mg digoxin bound per vial) wasadministered (time=T₀) in a composition of 3 vials digoxin immune Fabfor a total digoxin binding capacity of 1.5 mg. The composition wasadministered via an intravenous bolus infusion over thirty (30) minutes.Intravenously administered fluids were standardized to 125 cc/hr.

Urine output from admission to time of digoxin immune Fab (ovine)infusion (5 hours) was 300 cc (60 cc/hr). Over the first 6 hours postinitial administration of digoxin immune Fab, the average urinary outputincreased to 70 cc/hr with blood pressure readings of 148 to 162systolic and 104 to 111 diastolic. Due to insufficient response andcontinuing elevated blood pressure, at 6 hours after the initial dosage(T₆), a second dose was administered based upon a serum digoxinconcentration of 0.8 ng/mL (concentration believed to be present due toseverity of symptoms, but not quantified), resulting in a composition of6 vials for a total of 3.0 mg digoxin binding capacity. Afteradministration of the second dose, urine output increased to an averageof 140 cc/hr. The next dose (T₁₂) was omitted due to sustained clinicalimprovement. Doses were administered at approximately T₁₈ and T₂₄, eachtotaling 3.0 mg digoxin binding capacity. Doses were administered atapproximately T₃₀ and T₃₆, but due to clinical improvement the doseswere each decreased to 1.5 mg digoxin binding capacity. The final dosewas given approximately 12 hours prior to the time of anticipateddelivery. During the period of digoxin antibody dosing, a diuresis of7426 cc occurred for an average output of 177 cc/hr. The total intake ofintravenous fluids during the same time period was 5923 cc. The urinequalitative exam at T₁₀ revealed negative proteinuria with a specificgravity of 1.011. Urine osmolality at T₄₀ was 125 (normal 500 to 800mosm). The serum creatinine level decreased to 0.7 with the remainder ofelectrolytes remaining normal. Serial blood pressure measurements duringthe period from the doubling of the dose until deliver ranged from130s-140s systolic, 80s-90s diastolic. From the time of admission until12 hours following delivery, no antihypertensives were required. Theedema resolved in the face and upper extremities, with significantdecreases in the lower extremity edema to 1+. Deep tendon reflexesimproved to 1+ with no clonus.

Fetal assessment during this time showed no changes in thecardiotocograph. Ultrasound examination every 6 hours revealed areassuring biophysical profile score of 8 of 10. Doppler flow assessmentof the umbilical artery every 6 hours revealed a decreasingsystolic/diastolic ratio with an increasing diastolic component. The S/Dratio at T₁₅ was 3.2 with a RI of 0.69, suggesting that fetalhemodynamics were improved.

Cesarean delivery was performed at 48 hours post-administration ofcorticosteroids secondary to a breech presentation. The delivery wasattended by NICU and resulted in a live birth of a female child. Apgarscores were 7 and 8 at one and five minutes, respectively. The neonatedid not require any oxygen support and was admitted to the neonatalintensive care unit secondary to prematurity size of 1290 grams. Theneonate did not require oxygen during hospitalization and had no sepsisor intraventricular hemorrhage. She was discharged home on the 31st dayof life.

The maternal postoperative course was complicated by elevated bloodpressures of 140s-160s systolic and diastolic readings in the 100s. Thepatient was started on metoprolol 100 mg twice daily. She was dischargedhome on postoperative day #4 with stable blood pressures controlled withmetoprolol. All follow up laboratory tests remained within normallimits. The neonate had no adverse sequelae and was discharged home onday of life #31. A medical history was obtained from the mother 22months following delivery. The mother reported no chronic healthproblems, was not on any medications and had no hospitalizations sincedelivery. The child was evaluated by a pediatrician at 19 months of age.The pediatrician reported the child to be well, of normal growth andnormal development.

Biological Example #2

A 19 year old 74 Kg gravida 1, para 0 female at 29 weeks gestational agewas transferred to the tertiary care center due to elevated bloodpressure, dizziness, and excessive body edema most pronounced on face,hands, feet (2+). The patient was experiencing headache, blurred vision,epigastric pain, and shortness of breath. Blood pressure was reported as140-160s 80-90s, with a recording of 143/90 and a pulse of 61 atadmission. A grade 2 3/6 murmur with click was noted by the maternalfetal medicine (MFM) specialist. Past medical history was positive forspina bifida with no neurologic impairment. The family history waspositive for birth defects (heart disease and deafness), Grave'sdisease, and hypertension. The patient had experienced no problemsduring pregnancy prior to the day of admission. The patient was admittedfor surveillance and work-up of preeclampsia and steroid administration.She was given oxygen and intravenous fluids.

Ultrasound evaluation of fetus revealed fetal heart tones (FHTs) in the150s with acceleration. Fetal size was consistent with given dates.Doppler blood flow studies preinfusion revealed an S/D ratio of 4.84 andan RI of 0.79. Corticosteroid (betamethasone) was administered toaccelerate fetal lung maturity. A cardiology consult was obtained due tomaternal bradycardia (HR 50-66) and heart murmur. EKG showed sinusbradycardia and shortened PR interval. Echocardiography revealed leftventricular hypertrophy with minimal pericardial effusion and sinusbradycardia with no ectopy. Cardiology cleared the patient for surgery.

In the first 24 hours following admission the patient experiencedoliguria. The initial 24 hour urine resulted in 400 ml total volume andrevealed 16.5 gm of protein. Due to the oliguria, a composition of 6vials (3 mgs digoxin binding capacity) of digoxin immune Fab wereadministered as an intravenous bolus over thirty (30) minutes, basedupon a serum “digoxin” level of 8.0 ng/mL (believed to be present butnot quantified). A second dose of 6 vials was administered approximately15 hours after the initial dose. Urinary output (UOP) was noted toimprove with an increase to 800 ml for the next 24 hour urine with inprotein to 11.8 gm. Doppler blood flow (umbilical artery) improved witha decrease in the S/D ratio to 2.87.

A cesarean section and appendectomy were performed approximately 48hours post initial administration of steroids. The surgery was toleratedwell, and the post-operative course was uneventful with the exception ofleukocytosis, which was possibly attributed to the steroidadministration.

The patient delivered a viable female infant, 1476 gm with Apgar scoresof 7 and 9. The baby was transferred to the NICU in stable condition.She did not experience Respiratory Distress Syndrome, sepsis, orintraventricular hemorrhage. Her respiratory diagnosis was TransientTachypnea of the Neonate and she required CPAP×2 days and oxygen for atotal of 3 days. She was discharged home on day 28 of life.

The mother received prophylactic magnesium sulfate for 24 hours postdelivery. She was discharged on postpartum day 4 on vitamins and painmedication. The mother and child were evaluated 18 months afterdelivery. The mother indicated she had no health problems, nohospitalizations since delivery and in not on medication other thanbirth control. The infant was examined by a pediatrician who reportedthe infant to be physically and developmentally normal and thriving.

Biological Example #3

A 20 year old 91 Kg gravida 1, para 0 female presented to her localobstetrician at 31 weeks with a 5 lb weight gain, blood pressure222/112, and 3+ proteinuria. Prenatal care to this point wasuncomplicated although she did have trichomonas early in the pregnancy.The family history is pertinent in that the patient's mother had chronichypertension and had preeclampsia with all three of her pregnancies. Thepatient was hospitalized for stabilization and delivery. A maternalfetal medicine consult was obtained at which time the patient reportedscotomata and headache. There was no bleeding, leakage, contractions,right upper quadrant pain, or pre-ictal symptoms. There was 1+ pittingedema in the lower extremities with some edema in the lower sacral area.Clinical lab was unremarkable with the exception of 3+ proteinuria and aslightly elevated LDH. Good fetal movement was noted. On ultrasound, thefetus was vertex, anterior grade II placenta. Fetal size was consistentwith the stated age of 31 weeks with an estimated weight of 1499-1570grams. Doppler exam showed absent diastolic flow in the umbilical arterywith compensatory changes in the middle cerebral arteries. Resistanceindex was 0.78 with an S/D ratio of 4.7. Amniotic fluid index was about4 cm. The biophysical profile was 6 out of 10 with no decelerations,decreased variability, no contractions, and good movement and tone. Thediagnoses were severe preeclampsia, abnormal Doppler flow with absentdiastolic flow, oligohydramnios, and hypertensive urgency.

Upon admission magnesium sulfate was initiated. Within three hours ofadmission the patient was also on labetalol via continuous IV and hadreceived hydralazine 5 mg IV×2. Blood pressure was in the 140-160/100range following antihypertensive treatment. Corticosteroid(betamethasone) was administered to enhance pulmonary maturation of thefetus.

Because of insufficient antihypertensive response and diminished/absentfetal blood flow, digoxin immune Fab was administered. An initial dose(time=T₀) was determined using a serum “digoxin” concentration of 0.9ng/mL (believed to be present but not quantified), resulting in acomposition of 8 vials (0.5 mg digoxin bound per vial) totaling 4 mgdigoxin binding capacity. The composition was administered byintravenous bolus infusion over thirty (30) minutes. A second dosage(approximately T₂₈) and third dosage (approximately T₄₃), each of 4vials (2 mg total digoxin binding capacity per dose), were administeredbased upon a serum “digoxin” concentration of 4.5 ng/mL (believed to bepresent, but not quantified). The patient's symptoms improved overnightwith BPs 130s-150s/70s-90s. Urinary output increased and edemadecreased. Creatinine clearance (CrCl), measured on two occasions,increased from 147 on the second hospital day to 169 on the thirdhospital day. Fetal testing was reassuring. On repeat Doppler, diastolicflow was noted.

At approximately 48 hours post initial administration of steroid a 1520gram viable male infant was delivered via cesarean section. Apgar scoreswere 6 and 9. The infant's respiratory diagnosis was ProlongedTransition and he initially required no respiratory support. On day 4the infant developed clinical sepsis and necrotizing enterocolitis (NEC)and required surgery to repair an ileal perforation. He was on theventilator for 3 days post op, then weaned to room air for the remainderof his hospitalization. He experienced a Grade I intraventricularhemorrhage. He was discharged home at 64 days of age.

Following delivery the patient's blood pressure remained elevated140s-150s systolic, 80s-100s diastolic with labetalol. Nifedipine wasinitiated in attempt to wean the patient from labetalol. Otherwise, thepatient was progressing well. She was discharged home on post op day 4.At 18 months post delivery, a medical history was obtained from themother and the child was examined by a neonatologist and children'sphysical therapist. The mother reported no chronic health complicationsand no hospitalizations since delivery; she was not on any medications.The neonatologist found no abnormalities with the exception ofrhinorrhea and an abdominal scar. He described the child as healthy andthriving. The physical therapist found the developmental exam to benormal with motor level and other levels somewhat advanced for adjustedage. The child no longer requires follow-up with pediatric surgeon forileal perforation and has no dietary limitations.

Biological Example #4

A 29 year old 123 Kg gravida 3, para 2 female was air evacuated from aremote non-tertiary hospital to a tertiary care center afterexperiencing two seizures and amaurosis fugax (complete blindness). Thefirst seizure occurred at home and the second was witnessed byparamedics. Pregnancy history was notable for preeclampsia in theimmediate preceding pregnancy. This pregnancy was complicated bycholelithiasis and elevated BP for one week. Past medical and surgicalhistories were non-contributory. Family history was pertinent in thather mother has epilepsy. The patient was transported on magnesiumsulfate.

On admission to the tertiary center the patient's treatment continuedwith intravenous magnesium sulfate and administration of betamethasoneto improve neonatal respiratory outcomes. She was alert and oriented ×3and was experiencing headache, blurred vision and diplopia. Bloodpressure was in the 150s/90s. Urine protein was reported as 3+ at thereferring hospital. Physical exam was notable for swelling in the handsand face as well as 3+ pitting edema in the extremities with 4+ deeptendon reflexes and 1 beat of clonus. Admission 24-hour urine revealed acreatinine clearance of 98.2 with 1590 mg of protein. The patient'scondition was considered critical. She required oxygen administrationand placement of a central venous line. A one-time dose of labetalol 20mg was administered IV push in response to a BP of 162/109.

Primarily because of low urinary output, digoxin immune Fab wasinitiated, based upon a serum “digoxin” level of 4.0 ng/mL (believed tobe present, but not quantified), a composition of 5 vials (0.5 mgdigoxin binding capacity per vial) was administered by bolus infusionover thirty (30) minutes. The 5 vial dosage was repeated every six hoursfor 8 additional doses. A total antibody composition comprisingapproximately 22.5 mgs digoxin binding capacity was administered overthe course of treatment. During the hospital course the patient'sclinical condition improved. Clinical lab results improved and BP wasrelatively stable. Doppler flow studies showed improvement. Although nopre-infusion values are available, the S/D ratio improved from 4.1 to2.9, respectively, at 48 hours and 108 hours post initial antibodyinjection. It was decided to delay delivery.

The magnesium sulfate and digoxin immune Fab were discontinued followingthe eighth dose and the patient remained stable for approximately 24hours after which there was an increase in blood pressure. A ninth doseof 5 vials of digoxin immune Fab was administered with a marginaldecrease in BP and an increase in urinary output.

Five days following admission the patient delivered via cesarean sectiona 30 3/7 GA, 1181 gram male infant with Apgar scores of 8 and 9. Theinfant was transported on room air to the NICU due to prematurity.Despite his prematurity and small size, the infant did not experienceRespiratory Distress Syndrome and did not require ventilatory support.His respiratory diagnosis was pulmonary insufficiency of prematurity andhe required nasal CPAP for 2 days and oxygen-therapy for a total of 11days. A patent ductus arteriosus was successfully closed withindomethacin. The infant experienced no sepsis or intracranialhemorrhage. He was discharged home on an apnea monitor at 36 days ofage.

The maternal postpartum course was uneventful. Blood pressure remainedelevated and the patient was treated with oral antihypertensives. Themother was discharged on the 8^(th) hospital day with routinemedications as well as labetalol 100 mg bid orally. Both the mother andchild received follow-up evaluations 2 months post delivery. The motherreported no current medical problems and no hospitalizations sincedelivery. She was not on any medications. The 2 month old infant wasexamined by a neonatologist and children's physical therapist. Thephysical exam was normal. The infant had emesis and apnea associatedwith feeds and this was attributed due to overfeeding. The neonatologistrecommended a volume decrease. The physical therapy exam was normal.

Biological Example #5

A 17 year old 62 Kg gravida 1, para 0 female at 34½ weeks (33 weeks byultrasound) was found unresponsive, presumably postictal, and wasbrought to the local emergency room. She subsequently was witnessedundergoing generalized tonic-clonic seizures. Blood pressures weresignificantly elevated (160s/100s). Laboratory values on presentationincluded a uric acid of 13, dipstick urine protein of 3+ and serumcreatinine of 1.4. The patient was combative and unresponsive tocommands. There were multiple small lacerations, abrasions, andcontusions to the face and extremities. Past medical history wasnegative. The patient was treated with midazolam, hydralazine andlabetalol.

The patient was then transported to a tertiary care setting. On arrivalthe patient continued to exhibit combativeness and altered mentalstatus. Blood pressure was 151/107. Deep tendon reflexes were absent.Vaginal bleeding was noted along with uterine tetany suggestive ofabruption. Uterine activity was noted to be consistent with ahyperstimulatory pattern. The fetus was exhibiting severe variabledecelerations with decreased variability. The patient was taken to theoperating room where she had a cesarean section under generalanesthesia. There was 30% abruption of the placenta. A 2104 gram maleinfant was delivered with Apgar scores of 6, 8, and 9. The infant wastaken to intensive care in stable condition.

The patient was treated postoperatively with intravenous magnesiumsulfate. During the postpartum period she remained combative withaltered mental status for 3 days. A neurologic consult was obtained. Thepatient was bent in the fetal position, and was arousable to touch butwould moan and flail extremities. She was uncommunicative and unable tofollow commands. A CT scan of the brain revealed findings consistentwith hypertensive encephalopathy related to eclampsia. Hydralazine wasgiven for BP and phenergan and meperidine for pain. A propofol drip,midazolam drip and lorazepam were administered for agitation/sedation.The midazolam drip and restraints were employed throughout most of theperiod of postpartum encephalopathy. Blood pressure remained elevated,140-150s/90s. Follow-up neurologic assessments revealed little change.

Due to continued symptoms of hypertensive encephalopathy, digoxin immuneFab was administered based upon a level of 0.6 ng/mL serum “digoxin”concentration, resulting in a composition of 4 vials for a total of 2.0mgs digoxin binding capacity. The composition was administered byintravenous bolus infusion over thirty (30) minutes. Within 5 minutes ofinfusing digoxin immune Fab the patient became responsive to commandsand verbally communicative, with marked improvement. She was alert andresponsive, and was asking appropriately for her infant and forsomething to eat. The physician noted that the eclampsia post-ictalresolved 5 minutes after administration of antibody. Sedatives wereweaned. On the day following antibody administration the patient wastransferred out of ICU. On the sixth postpartum day the patient wasdischarged on labetalol for hypertension, and ibuprofen andoxycodone/acetaminophen for pain.

Biological Example #6

Endogenous digoxin-like factors have been reported by some investigatorsto be elevated in preeclampsia. If present, then the site of action forthese inhibitors could be Na⁺/K⁺ ATPase. To test this possibility,erythrocyte Na⁺/K⁺ ATPase activity was determined in pregnanciescomplicated by severe preeclampsia, in normotensive pregnancies and inhealthy non-pregnant women. Whole blood was obtained from 12 subjects ineach of the three categories. Erythrocytes were isolated and placed incocktails of HEPES buffer solution at a 50% hematocrit and incubatedwith ⁸⁶Rubidium (Rb⁺), a potassium analog, for three hours. Na⁺/K⁺ATPase activity was determined by the uptake of the Rb⁺, in erythrocytesand expressed as nmol/hr/10⁶ cells. Erythrocyte Na⁺/K⁺ ATPase activitywas significantly increased in normotensive pregnancies as compared tonormotensive non-pregnant subjects (81.4±8.2 nmol/hour/10⁶ cells, vs61.1±7.4 nmol/hour/10⁶ cells, mean±S.D., p<0.01). Erythrocyte Na⁺/K⁺ATPase activity was significantly decreased in severe preeclampticpatients as compared to normotensive pregnancies (46.4±14.1nmol/hour/10⁶ cells).

The effect of digoxin antibody on erythrocyte Na⁺/K⁺ ATPase activity insevere preeclampsia was also evaluated. Whole blood was obtained from 12normotensive pregnant women and 12 severe preeclamptic women.Erythrocytes were isolated and placed in cocktails of HEPES buffersolution at a 50% hematocrit and incubated with Rb⁺. Digoxin antibody inthe form of DIGIBIND® was administered to 6 preeclamptic patients andnormal saline placebo was administered to 6 preeclamptic patients at thetime of umbilical cord clamping. DIGIBIND® was administered in a 50 ccnormal saline bag using a dose calculated by the formula of anendogenous digitalis like factor of 0.2 ng/mL and compositionsappropriate for each patient's weight were selected from Table III.

Na⁺/K⁺ ATPase activity was determined at baseline (delivery), 6, 12 and24 hours by the uptake of Rb⁺ in erythrocytes and expressed asnmol/hr/10⁶ cells. Mean Na⁺/K⁺ ATPase activity increased toward normalcontrols in both the DIGIBIND® and placebo treated preeclamptic samples;however, for the placebo treated preeclamptic samples, the increase inactivity appeared to be more gradual and modest. (See Table IV below).

TABLE IV Digoxin Antibody Effect on Sodium/Potassium ATPase Activity*Group Baseline 6 hours 12 hours 24 hours Normal Control 82.2 ± 8.2 79.0± 11.9 82.6 ± 8.4 86.1 ± 14.3 Samples (N = 12) Digibind ®  48.4 ± 18.463.2 ± 19.3 62.0 ± 9.1 73.8 ± 24.3 Treated Samples (N = 6) Placebo 44.0± 9.3 51.8 ± 14.5  53.8 ± 12.0 59.5 ± 12.4 Treated Samples (N = 6)*Activity expressed as ⁸⁶Rb⁺ uptake nmol/hr/10⁶ RBC

Biological Example #7

Digoxin antibody (specifically DIGIBIND® or DIGIFAB™, referred to as“DFAB”) will be evaluated in a clinical trial. This study will be adouble-blind, placebo-controlled, randomized trial of the efficacy ofdigoxin antibody for the treatment of 26 patients with severepreeclampsia. Patients must meet both diagnostic criteria forpreeclampsia and meet at least criteria for severe preeclampsia, asdefined by the American College of Obstetrics and Gynecology. Noparticipant will have received digoxin. After obtaining informedconsent, patients will be randomized 1:1 to receive either digoxinantibody or placebo (normal saline) over 48 hours (8 total singleapplication doses). A total of 13 patients will receive DFAB and 13patients will receive placebo (normal saline). The DFAB doses (mgdigoxin binding capacity) will be determined using a serum “digoxin”concentration of 0.6 ng/mL and compositions appropriate for eachpatient's weight will be selected from Table III. Compositions will beadministered approximately every six hours. The contents of individualvials should be first dissolved with 4 ml of sterile water forinjection, by gentle mixing, to give a clear, colorless solution. Thereconstituted vials should be collectively diluted in normal saline to atotal of 50 cc. The study drug should be administered as an intravenousbolus injection over a 10 minute period. Patients will be assessed atscreen (baseline) and monitored over the 48 hour period of study drugadministration.

Blood pressure measurements will be obtained hourly. A significantdifference in mean arterial pressure is expected within four hours afteradministration of each application dose with significant lowering in theDFAB group from baseline, and the trend is expected to continue throughat least 6 hours. Patients will be allowed antihypertensive therapy ifthe clinician determines such therapy is necessary. It is expected thatthere will be a statistically significant difference between the groupswith 46% of the patients in the placebo group expected to requireantihypertensives and none of the patients in the DFAB group areexpected to require antihypertensives (p<0.01).

Creatinine clearance will be calculated by the standard formula:Clearance (ml/min)=U_(Cr)×V/P_(Cr) where U_(Cr)=urine creatinineconcentration in mg/dL; V=urine volume in mL/min.; and P_(Cr)=plasmacreatinine in mg/dL. A statistically significant increase in 24 hourcreatinine clearance from baseline is also expected for the DFAB group(DFAB 147±23 mL/min.; placebo 95±25 ml/min., p≦0.05). However, a 30%increase in creatinine clearance from baseline (representingapproximately 30 mL/min.) in the DFAB group versus the placebo groupwould be considered a clinically beneficial change.

A clinically beneficial or statistically significant decrease inproteinuria is expected during study drug administration. If baseline isbetween >1+ and <3+ on urine dipstick (i.e., >300 mg but <500 mg/24 hr),it will be considered clinically beneficial for a decrease from baselineto negative proteinuria. A statistically significant decrease isexpected for baseline proteinuria≧3+ (≧500 mg/24 hr) if reduction frombaseline is ≧33% (p≦0.05). No adverse events are expected in anypatient.

Biological Example #8

A 19 year old, 70 kg nulliparous, gravida 2, with a familial history ofpreeclampsia would present to the obstetrician at 28 weeks' gestationfor routine prenatal exam. Prior blood pressures would be in the rangeof 90-110 systolic, 70s diastolic. At 28 weeks, blood pressure would be130/90 and urine dipstick of <1. Physical exam suggested fetal growthrestriction so Doppler blood flow studies would be indicated. Dopplerwould reveal an S/D ratio of 4.0 and an RI of 0.78, and intrauterinegrowth restriction (fetal size consistent with 25 week gestation). Serum“digoxin” concentration would be undetectable. Due to increasing bloodpressures, reduced umbilical artery flow, and intrauterine growthrestriction in a multifetal gestation and with a history of preeclampsia(patient's mother and sister), the patient would be indicated fortreatment with digoxin immune Fab. The patient would initially beindicated for an intramuscular injection of 40 mgs digoxin immune Fab(DIGIFAB™) having a total of 0.5 mgs digoxin binding capacity. Thepatient would initially be administered 40 mg (0.5 mg digoxin bindingcapacity) digoxin immune Fab (DIGIFAB™) by intramuscular injection atleast once each week and instructed to self-monitor and report dailyblood pressure measurements. Frequency and/or doses of digoxin immuneFab treatment would be increased if blood pressure measurementsincrease. Patient would be evaluated for blood pressure and proteinuriaon weekly follow-up visits. On follow-up visits from 29 weeks to 35weeks, blood pressure would be in 120s-130s systolic, 70s-80s diastolic,doppler blood flow of umbilical artery would show improvement with a S/Dratio of 2.5 and reassuring biophysical profile, dipstick<1 and normalurinary output and normal serum creatinine levels.

If at 36 weeks, the patient's weight is 82 Kg, with a BP 140/90,proteinuria is observed 1+, and/or umbilical doppler indicates anincrease in umbilical artery constriction (e.g., S/D of 4.5) or absentdiastolic flow, then the patient would be admitted to the hospital withan anticipated delivery in approximately 48 hours. Bethamethasone (12mgs intramuscular) would be administered upon hospital admission andapproximately 12 hours later. Approximately 60 minutes afterbethamethasone injection, the patient would be given a loading dose of 4vials (160 mgs, total 2 mgs digoxin binding capacity) digoxin immune Fab(DIGIFAB™) by bolus infusion. Total intravenous fluids would bestandardized to 125 cc/hr and digoxin immune Fab would be continued asan infusion at a rate of 4 mg per hour. During DIGIFAB™ administration,mean BPs are expected to be in the 120s-130s systolic, 70s-80s diastolicand protein dipstick is expected to be <1. Umbilical artery flow isexpected to show improvement (e.g., S/D of 2.5) with an increaseddiastolic component. A second dose of 12 mgs bethamethasone would beadministered approximately 24 hours after admission, and delivery wouldbe performed approximately 24 hours after the last dose ofbethamethasone. Two viable neonates would be delivered having Apgars of7 and 8.

In addition to object, features and advantages in the embodiments andexamples described above, other objects, features and advantages of thepresent invention will be apparent to those skilled in the art. Fromtime to time various professional medical organizations may modify themedical criteria for diagnosis of preeclampsia and eclampsia, and thepresent invention is not limited to the specific clinical symptoms,indications or diagnostic factors described herein.

While preferred examples and steps of the present invention have beenillustrated and described, this has been by way of illustration and theinvention should not be limited except as required by the scope of theappended claims and their equivalents.

TABLE V Summary Biological Examples Nos. 1-5* Preeclampsia Indicationfor Eclampsia or Patient # Treatment IUGR Weight Dose Regimen ResponseEffective 1 Hypertension; Eclampsia/ 70 Kg T₀ - 3 vials ↓BP Yes ↓ urineoutput; at risk for IUGR T_(12, 18, 24) - 6 vials ↑ urinary output ↓fetal blood T_(30, 36) - 3 vials ↓ proteinuria flow Dose in first 30hrs: ↓ edema 0.171 mg/kg ↑ fetal blood flow ↓ serum creatinine ↓ deeptendon reflexes 2 Oliguria; Severe 74 Kg T₀ - 6 vials ↑ urinary outputYes ↓ fetal blood preeclampsia/ T₁₅ - 6 vials ↑ fetal blood flow flow atrisk for IUGR Dose in first 30 hrs: 0.081 mg/kg 3 Hypertension; Severe91 Kg T₀ - 8 vials ↓ BP Yes ↓ (or absent) preeclampsia/at T₂₈ - 4 vials↓ edema fetal blood flow risk for IUGR T₄₃ - 4 vials ↑ fetal blood flowDose in first 30 hrs: ↑ creatinine 0.066 mg/kg clearance 4 ↓urinaryoutput; Eclampsia/ 123 Kg  T_(0, 6, 12, 18,24 30, 36, 42) - ↓BP Yes ↓fetal blood At risk for IUGR 5 vials ↑urinary output flow T₆₆ - 5 vials↑fetal blood flow Dose in first 30 hrs: 0.122 mg/kg 5 Hypertensive;eclampsia with 62 Kg 4 vials restoration of Yes Eclamptic encephalopathy0.032 mg/kg mental status Encephalopathy *Biological Examples #1-5represent individual case reports. Results of cellular assays (#6),prophetic clinical trials (#7) and prophetic individual case examplesare not included in this Table V.

What is claimed is:
 1. A method of extending pregnancy in a gravid humanpatient by reducing endogenous inhibition of Na+, K+ ATPase activity ina cell of the gravid human patient exhibiting at least one symptom ofgestational hypertension, preeclampsia, eclampsia, or intrauterinegrowth restriction comprising administration of a dosage of digoxinantibody to the gravid human patient, wherein the dosage is effective toreduce endogenous inhibition of Na+, K+ ATPase activity and extendpregnancy in the patient.
 2. The method of claim 1, wherein the patientexhibits proteinuria levels greater than 300 mg over an interval of 24hours prior to administration.
 3. The method of claim 2, comprisingadministration of subsequent doses of digoxin antibody at least untilthe levels of proteinuria in the gravid human patient decrease afteradministration.
 4. The method of claim 1, wherein the at least onesymptom includes hypertension greater than 140 mm Hg systolic or 90 mmHg diastolic and proteinuria greater than 300 mg per 24 hours.
 5. Themethod of claim 1, wherein the at least one symptom includeshypertension greater than 160 mm Hg systolic or 110 mm Hg diastolic andproteinuria greater than 500 mg per 24 hours.
 6. The method of claim 1,wherein the at least one symptom includes hypertension greater than 140mm Hg systolic or 90 mm Hg diastolic and proteinuria greater than 1+ ontwo random urine dipstick samples collected at least 4 hours apart. 7.The method of claim 1, wherein the at least one symptom includeshypertension greater than 160 mm Hg systolic or 110 mm Hg diastolic andproteinuria greater than 3+ on two random urine dipstick samplescollected at least 4 hours apart.
 8. The method of claim 1, wherein thedosage is administered over a period of six hours or less.
 9. The methodof claim 8 comprising administration of subsequent dosages of digoxinimmune Fab.
 10. The method of claim 1, wherein the method furthercomprises administering a therapeutically effective amount ofcorticosteroid.
 11. The method of claim 1, wherein the method furthercomprises administering a therapeutically effective amount of anantihypertensive drug.
 12. The method of claim 11, wherein theantihypertensive drug is labetalol, altenolol, nifedipine, 1-methyldopaor hydralazine.
 13. The method of claim 1, wherein the method furthercomprises administering a therapeutically effective amount of magnesiumsulfate or phenytoin.
 14. The method of claim 1, wherein the digoxinimmune Fab is ovine digoxin immune Fab.
 15. A method of extendingpregnancy in a gravid human patient by increasing Na+, K+ ATPaseactivity in a cell of the gravid human patient exhibiting endogenousinhibition of Na+, K+ ATPase, comprising administration of a dosage ofdigoxin antibody to the gravid human patient, wherein the dosage iseffective to increase Na+, K+ ATPase activity and extend pregnancy inthe human patient.
 16. The method of claim 15, wherein the patientexhibits proteinuria levels greater than 300 mg over an interval of 24hours prior to administration.
 17. The method of claim 16, comprisingadministration of subsequent doses of digoxin antibody at least untilthe levels of proteinuria in the gravid human patient decrease afteradministration.
 18. The method of claim 15, wherein the at least onesymptom includes hypertension greater than 140 mm Hg systolic or 90 mmHg diastolic and proteinuria greater than 300 mg per 24 hours.
 19. Themethod of claim 15, wherein the at least one symptom includeshypertension greater than 160 mm Hg systolic or 110 mm Hg diastolic andproteinuria greater than 500 mg per 24 hours.
 20. The method of claim15, wherein the at least one symptom includes hypertension greater than140 mm Hg systolic or 90 mm Hg diastolic and proteinuria greater than 1+on two random urine dipstick samples collected at least 4 hours apart.21. The method of claim 15, wherein the at least one symptom includeshypertension greater than 160 mm Hg systolic or 110 mm Hg diastolic andproteinuria greater than 3+ on two random urine dipstick samplescollected at least 4 hours apart.
 22. The method of claim 15, whereinthe dosage is administered over a period of six hours or less.
 23. Themethod of claim 22, comprising administration of subsequent dosages ofdigoxin immune Fab.
 24. The method of claim 15, wherein the methodfurther comprises administering a therapeutically effective amount ofcorticosteroid.
 25. The method of claim 15, wherein the method furthercomprises administering a therapeutically effective amount of anantihypertensive drug.
 26. The method of claim 25, wherein theantihypertensive drug is labetalol, altenolol, nifedipine, 1-methyldopaor hydralazine.
 27. The method of claim 15, wherein the method furthercomprises administering a therapeutically effective amount of magnesiumsulfate or phenytoin.
 28. The method of claim 15, wherein the digoxinimmune Fab is ovine digoxin immune Fab.